Alcian Blue Binding Research Articles

Achillea millefolium Essential Oil Mitigates Peptic Ulcer in Rats through Nrf2/HO-1 Pathway.

Extreme ethanol ingestion is associated with developing gastric ulcers. Achillea millefolium (yarrow) is one of the most commonly used herbs with numerous proven pharmacological actions. The goal of the hereby investigation is to explore the gastroprotective action of yarrow essential oil against ethanol-induced gastric ulcers and to reveal the unexplored mechanisms. Rats were distributed into five groups (n = 6); the control group administered 10% Tween 20, orally, for two weeks; the ethanol group administered absolute ethanol (5 mL/kg) to prompt gastric ulcer on the last day of the experiment. Yarrow essential oil 100 or 200 mg/kg + ethanol groups pretreated with yarrow oil (100 or 200 mg/kg, respectively), orally, for two weeks prior to gastric ulcer induction by absolute ethanol. Lanso + ethanol group administered 20 mg/kg lansoprazole, orally, for two weeks prior to gastric ulcer induction by ethanol. Results of the current study showed that ethanol caused several macroscopic and microscopic alterations, amplified lipid peroxidation, pro-inflammatory cytokines, and apoptotic markers, as well as diminished PGE2, NO, and antioxidant enzyme activities. On the other hand, animals pretreated with yarrow essential oil exhibited fewer macroscopic and microscopic modifications, reduced ulcer surface, and increased Alcian blue binding capacity, pH, and pepsin activity. In addition, yarrow essential oil groups exhibited reduced pro-inflammatory cytokines, apoptotic markers, and MDA, restored the PGE2 and NO levels, and recovered the antioxidant enzyme activities. Ethanol escalated Nrf2 and HO-1 expressions, whereas pretreatment of yarrow essential oil caused further intensification in Nrf2 and HO-1. To conclude, the current study suggested yarrow essential oil as a gastroprotective agent against ethanol-induced gastric lesions. This gastroprotective effect could be related to the antioxidant, anti-inflammatory, and anti-apoptotic actions of the essential oil through the instigation of the Nrf2/HO-1 pathway.

Antioxidant and Gastroprotective Activity of Suaeda fruticosa Forssk. Ex J.F.Gmel.

Suaeda fruticosa Forssk. Ex J.F.Gmel is traditionally used for inflammatory and digestive disorders, as a carminative, and for diarrhea. This plant is widely distributed in Asia, Africa, and the Mediterranean region. Aqueous methanolic extract of S. fruticosa (Sf.Cr) was prepared and screened for phytoconstituents through qualitative and GC-MS analysis. Quantification of total phenolic and flavonoid contents was performed, while antioxidant capacity was determined by DPPH, CUPRAC, FRAP, and ABTS assays. The gastroprotective activity was assessed in an ethanol-induced ulcer model. Gastric secretory parameters and macroscopic ulcerated lesions were analyzed and scored for ulcer severity. After scoring, histopathology was performed, and gastric mucus contents were determined. Oral pre-treatment of Sf.Cr demonstrated significant gastroprotection. The gastric ulcer severity score and ulcer index were reduced while the %-inhibition of ulcer was increased dose-dependently. The Sf.Cr significantly elevated the pH of gastric juice, while a decrease in total acidity and gastric juice volume was observed. Histopathology demonstrated less oedema and neutrophil infiltration in gastric mucosa of rats pre-treated with the Sf.Cr in comparison to ethanol-intoxicated animals. Furthermore, the gastric mucus contents were increased as determined by alcian blue binding. Sf.Cr showed marked gastroprotective activity, which can be attributed to antioxidant, antisecretory, and cytoprotective effects.

Brassica oleracea Prevents HCl/Ethanol-Induced Gastric Damages in Mice

Brassica oleracea var. capitata L. (cabbage) is a popular vegetable with a wide range of pharmacological activities that help to promote human health. The present study investigated the beneficial effects of B. oleracea var. capitata L. extract (BOE) on HCl/ethanol (H/E)-induced gastric damages in mice. Pre-administration of BOE (25–100 mg/kg) for 7 consecutive days significantly decreased macroscopically visible lesion on the gastric mucosa induced by H/E. In addition, results from hematoxylin and eosin-stained gastric tissue showed that BOE inhibited invaded percentage of lesion and prevented the reduction in mucosal thickness in peri-ulcerative region. BOE significantly alleviated the H/E-mediated decreases in Alcian blue binding, total hexose, sialic acid, and collagen in the gastric tissue, suggesting BOE attenuates the gastric damage via preserving the integrity of gastric mucus. Moreover, BOE significantly decreased histamine level in the plasma and reduced mRNA levels associated with secreting gastric acid. Furthermore, BOE inhibited myeloperoxidase activity and suppressed nuclear factor-κB mRNA and its dependent inflammatory genes expression induced by H/E. BOE also strengthened antioxidant enzyme activity, with a mitigating H/E-mediated increase in malondialdehyde level of the gastric tissue. Thus, these results suggest that BOE has the potential to protect the gastric tissue via inhibiting gastric acid secretion, inflammation, and oxidative stress.

Both Svp26 and Mnn6 are required for the efficient ER exit of Mnn4 in Saccharomyces cerevisiae.

Incorporation of membrane and secretory proteins into COPII vesicles are facilitated either by the direct interaction of cargo proteins with COPII coat proteins, or by ER exit adaptor proteins which mediate the interaction of cargo proteins with COPII coat proteins. Svp26 is one of the ER exit adaptor proteins in the yeast Saccharomyces cerevisiae. The ER exit of several type II membrane proteins have been reported to be facilitated by Svp26. We demonstrate here that the efficient incorporation of Mnn4, a type II membrane protein required for mannosyl phosphate transfer to glycoprotein-linked oligosaccharides, into COPII vesicles is also dependent on the function of Svp26. We show that Mnn4 is localized to the Golgi. In addition to Mnn4, Mnn6 is known to be also required for the transfer of mannosyl phosphate to the glycans. We show, by indirect immunofluorescence, that Mnn6 localizes to the ER. As in the case with Svp26, deletion of the MNN6 gene results in the accumulation of Mnn4 in ER. In vitro COPII vesicle budding assays show that Svp26 and Mnn6 facilitate the incorporation of Mnn4 into COPII vesicles. In contrast to Svp26, which is itself efficiently captured into the COPII vesicles, Mnn6 was not incorporated into the COPII vesicles. Mnn4 and Mnn6 have the DXD motif which is often found in the many glycosyltransferases and functions to coordinate a divalent cation essential for the reaction. Alcian blue dye binding assay shows that substitution of the first D in this motif present in Mnn4 by A impairs the Mnn4 function. In contrast, amino acid substitutions in DXD motifs present in Mnn6 did not affect the function of Mnn6. These results suggest that Mnn4 may be directly involved in the mannosyl phosphate transfer reaction.

Evidence of the gastroprotective and anti- Helicobacter pylori activities of β-mangostin isolated from Cratoxylum arborescens (vahl) blume.

Purposeβ-Mangostin (BM) from Cratoxylum arborescens demonstrated various pharmacological activities such as anticancer and anti-inflammatory. In this study, we aimed to investigate its antiulcer activity against ethanol ulcer model in rats.Materials and methodsBM was isolated from C. arborescens. Gastric acid output, ulcer index, gross evaluation, mucus production, histological evaluation using hematoxylin and eosin and periodic acid–Schiff staining and immunohistochemical localization for heat shock protein 70 (HSP70) and Bax proteins were investigated. Possible involvement of reduced glutathione, lipid peroxidation, prostaglandin E2, antioxidant enzymes, superoxide dismutase and catalase enzymes, radical scavenging, nonprotein sulfhydryl compounds, and anti-Helicobacter pylori were investigated.ResultsBM showed antisecretory activity against the pylorus ligature model. The pretreatment with BM protect gastric mucosa from ethanol damaging effect as seen by the improved gross and histological appearance. BM significantly reduced the ulcer area formation, the submucosal edema, and the leukocytes infiltration compared to the ulcer control. The compound showed intense periodic acid–Schiff staining to the gastric mucus layer and marked amount of alcian blue binding to free gastric mucus. BM significantly increased the gastric homogenate content of prostaglandin E2 glutathione, superoxide dismutase, catalase, and nonprotein sulfhydryl compounds. The compound inhibited the lipid peroxidation revealed by the reduced gastric content of malondialdehyde. Moreover, BM upregulate HSP70 expression and downregulate Bax expression. Furthermore, the compound showed interesting anti-H. pylori activity.ConclusionThus, it could be concluded that BM possesses gastroprotective activity, which could be attributed to the antisecretory, mucus production, antioxidant, HSP70, antiapoptotic, and anti-H. pylori mechanisms.

Widening and diversifying the proteome capture by combinatorial peptide ligand libraries via Alcian Blue dye binding.

Combinatorial peptide ligand libraries (CPLLs) tend to bind complex molecules such as dyes due to their aromatic, heterocyclic, hydrophobic, and ionic nature that may affect the protein capture specificity. In this experimental work Alcian Blue 8GX, a positively charged phthalocyanine dye well-known to bind to glycoproteins and to glucosaminoglycans, was adsorbed on a chemically modified CPLL solid phase, and the behavior of the resulting conjugate was then investigated. The control and dye-adsorbed beads were used to harvest the human urinary proteome at physiological pH, this resulting in a grand total of 1151 gene products identified after the capture. Although the Alcian Blue-modified CPLL incremented the total protein capture by 115 species, it particularly enriched some families among the harvested proteins, such as glycoproteins and nucleotide-binding proteins. This study teaches that it is possible, via the two combined harvest mechanisms, to drive the CPLL capture toward the enrichment of specific protein categories.

Pretreatment of Gymnema sylvestre revealed the protection against acetic acid-induced ulcerative colitis in rats

BackgroundOverproduction of free radicals and decreased antioxidant capacity are well-known risk factors for inflammatory bowel diseases. Gymnema sylvestre (GS) leaves extract is distinguished for its anti-diabetic, antioxidant and anti-inflammatory properties. Present study is designed to evaluate the preventative activities of GS against acetic acid (AA)-induced ulcerative colitis in Wistar rats.MethodsExperimentally ulcerative colitis (UC) was induced by AA in animals pretreated with three different doses of GS leaves extract (50, 100, 200 mg/kg/day) and a single dose of mesalazine (MES, 300 mg/kg/day) for seven days. Twenty four hours later, animals were sacrificed and the colonic tissues were collected. Colonic mucus content was determined using Alcian blue dye binding technique. Levels of thiobarbituric acid reactive substances (TBARS), total glutathione sulfhydryl group (T-GSH) and non-protein sulfhydryl group (NPSH) as well as the activity of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) were estimated in colon tissues. Colonic nucleic acids (DNA and RNA) and total protein (TP) concentrations were also determined. Levels of pro-inflammatory cytokines including interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) as well as prostaglandin E2 (PGE2) and nitric oxide (NO) were estimated in colonic tissues. The histopathological changes of the colonic tissues were also observed.ResultsIn AA administered group TBARS levels were increased, while colonic mucus content, T-GSH and NP-SH, SOD and CAT were reduced in colon. Pretreatment with GS inhibited TBARS elevation as well as mucus content, T-GSH and NP-SH reduction. Enzymatic activities of SOD and CAT were brought back to their normal levels in GS pretreated group. A significant reduction in DNA, RNA and TP levels was seen following AA administration and this inhibition was significantly eliminated by GS treatment. GS pretreatment also inhibited AA-induced elevation of pro-inflammatory cytokines, PGE2 and NO levels in colon. The apparent UC protection was further confirmed by the histopathological screening.ConclusionThe GS leaves extract showed significant amelioration of experimentally induced colitis, which may be attributed to its anti-inflammatory and antioxidant property.

The gastroprotective effect of oxycellulose pellets on gastric ulcers

Oxycellulose belongs to the wide range of pharmaceutically and medically used cellulose derivatives unique in its properties of biodegradability, non-toxicity, antioxidative activity, haemostasis supporting, tissue healing improvement, etc. Glycyrrhizin (Gly) represents the most prominent licorice triterpenoid glycoside, which is responsible for its pharmacological activity, and is closely linked to increasing levels of gastroprotective prostaglandins. In order to study the anti-ulcer activity of oxycellulose sodium and Gly, pellets containing only oxycellulose, or in combination with glycyrrhizin, were prepared using an extrusion/spheronization method. The physical properties and in vitro release rate of the prepared pellets were tested. Based on the obtained results, one sample prepared from oxycellulose only, and one containing glycyrrhizin were chosen for in vivo testing. The healing effect of the prepared pellet formulations was evaluated in the acetic acid induced gastric ulcers in rats. The improvement in gastric ulcer healing was documented for myeloperoxidase activity, alkaline phosphatase activity and gastric wall mucin levels using the Alcian blue binding capacity. The tested pellets significantly reduced myeloperoxidase and alkaline phosphatase activity, and increased gastric wall mucus levels compared to the non-treated group. The in vivo tests showed that oral administration of the prepared pellets accelerated the healing of gastric ulcers in rats.

Cartilage collagen damage in hip osteoarthritis similar to that seen in knee osteoarthritis; a case–control study of relationship between collagen, glycosaminoglycan and cartilage swelling

BackgroundIt remains to be shown whether OA shares molecular similarities between different joints in humans. This study provides evidence for similarities in cartilage molecular damage in osteoarthritic (OA) joints.MethodsArticular cartilage from osteoarthritic hip joints were analysed and compared to non-OA controls regarding collagen, glycosaminoglycan and water content. Femoral heads from 16 osteoarthritic (OA) and 20 reference patients were obtained from hip replacement surgery due to OA and femoral neck fracture, respectively. Cartilage histological changes were assessed by Mankin grading and denatured collagen type II immunostaining and cartilage was extracted by α-chymotrypsin. Hydroxyproline and Alcian blue binding assays were used to measure collagen and glycosaminoglycan (GAG) content, respectively.ResultsMankin and immunohistology scores were significantly higher in hip OA samples than in reference samples. Cartilage water content was 6% higher in OA samples than in references. 2.5 times more collagen was extracted from OA than from reference samples. There was a positive association between water content and percentage of extractable collagen pool (ECP) in both groups. The amounts of collagen per wet and dry weights did not differ statistically between OA and reference cartilage. % Extractable collagen was not related to collagen per dry weight in either group. However when collagen was expressed by wet weight there was a negative correlation between % extractable and collagen in OA cartilage. The amount of GAG per wet weight was similar in both groups but the amount of GAG per dry weight was higher in OA samples compared to reference samples, which suggests a capacity for GAG biosynthesis in hip OA cartilage. Neither of the studied parameters was related to age in either group.ConclusionsIncreased collagen extractability and water content in human hip cartilage is associated with OA pathology and can be observed at early stages of the degenerative hip OA process. Our results suggest a common degradative pathway of collagen in articular cartilage of different joints. Furthermore, the study suggests that biochemical changes precede more overt OA changes and that chondrocytes may have a capability to compensate molecular loss in the early phase of OA.

Protective effect of naringenin on acetic acid-induced ulcerative colitis in rats

To evaluate the ameliorative effect of naringenin (NG) during ulcerative colitis (UC) in rats. Rats were treated with three different doses (25, 50 and 100 mg/kg per day) of NG and a single dose of mesalazine (MES, 300 mg/kg per day) for seven days prior to ulcerative colitis induction by 4% acetic acid (AA). Twenty four hours after AA rectal administration, animals were scarified and the colonic tissues were dissected. Colonic mucus content was estimated using Alcian blue dye binding technique. In colon tissues, levels of total glutathione sulphadryls (T-GSH), non-protein sulphadryls (NP-SH) and thiobarbituric acid reactive substances (TBARS) were evaluated. The activities of the antioxidant enzymes, catalase (CAT) and superoxide dismutase (SOD) were measured. Concentrations of nucleic acids (DNA and RNA) and total protein were also estimated in colon tissues. Colonic levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), prostaglandin E2 (PGE2) and nitric oxide (NO) were estimated. In cross section of colitis tissue the histopathological changes were observed. Colonic mucus content was decreased in AA compared to controls (587.09 ± 65.59 mg/kg vs 941.78 ± 68.41 mg/kg, P < 0.001). AA administration markedly reduced T-GSH (5.25 ± 0.37 nmol/L vs 3.04 ± 0.24 nmol/L, P < 0.01), NP-SH (3.16 ± 0.04 nmol/L vs 2.16 ± 0.30 nmol/L, P < 0.01), CAT (6.77 ± 0.40 U/mg vs 3.04 ± 0.2 U/mg, P < 0.01) and SOD (3.10 ± 0.11 U/mg vs 1.77 ± 0.18 U/mg, P < 0.01) while TBARS, TNF-α, IL-1β, IL-6, PGE2 and NO levels (15.09 ± 3.84 nmol/L vs 59.90 ± 16.34 nmol/L, P < 0.01; 113.56 ± 1.91 pg/mg vs 134.24 ± 4.77 pg/mg, P < 0.01; 209.20 ± 36.38 pg/mg vs 422.19 ± 31.47 pg/mg, P < 0.01; 250.83 ± 25.09 pg/mg vs 638.58 ± 115.9 pg/mg, P < 0.01; 248.19 ± 36.98 pg/mg vs 541.74 ± 58.34 pg/mg, P < 0.01 and 81.26 ± 2.98 mmol/g vs 101.90 ± 10.73 mmol/g, P < 0.001) were increased in colon of rats with UC compared controls respectively.Naringenin supplementation, significantly and dose dependently increased the colonic mucus content. The elevated TBARS levels were significantly decreased (39.35 ± 5.86 nmol/L, P < 0.05; 26.74 ± 3.17 nmol/L, P < 0.01 nmol/L and 17.74 ± 2.69 nmol/L, P < 0.01) compared to AA (59.90 ± 16.34 nmol/L) group while the decreased levels of T-GSH and NP-SH and activities of CAT and SOD found increased by NG treatments in dose dependent manner. The decreased values of nucleic acids and total protein in AA group were also significantly (P < 0.01) increased in all three NG supplemented groups respectively. NG pretreatment inhibited the TNF-α levels (123.76 ± 3.76 pg/mg, 122.62 ± 3.41 pg/mg and 121.51 ± 2.61 pg/mg vs 134.24 ± 4.78 pg/mg, P < 0.05) compared to AA group, respectively. Interleukins, IL-1β and IL-6 levels were also decreased in NG50 + AA (314.37 ± 16.31 pg/mg and 292.58 ± 23.68 pg/mg, P < 0.05) and NG100 + AA (416.72 ± 49.62 pg/mg and 407.96 ± 43.87 pg/mg, P < 0.05) when compared to AA (352.46 ± 8.58 pg/mg and 638.58 ± 115.98 pg/mg) group. Similar decrease (P < 0.05) was seen in PGE2 and NO values when compared to AA group. The group pretreated with MES, as a reference drug, showed significant (P < 0.01) protection against the changes induced in colon tissue by AA administration respectively. In present study, NG produced antioxidant and anti-inflammatory effects demonstrating protective effect in inflammatory bowel disease.

Gastric antisecretory and antiulcer activity of bovine hemoglobin

To investigate gastric antisecretory and gastroprotective activity of bovine hemoglobin (B-Hb) in rats. Adult Albino-Wistar rats were divided into groups of 6 animals each. B-Hb in doses of 100, 300 and 900 mg/kg body weight was tested for gastric acid secretion and antiulcer activity. Gastric secretions were measured 6 h after pylorus ligation in rats pretreated with B-Hb. The acidity was measured by titrating gastric contents against 0.01 mol/L NaOH to pH 7. Indomethacin ulcers were produced by oral administration of 30 mg/kg bw in the rats pretreated with B-Hb one hour before indomethacin. Six hours after indomethacin stomach removed and ulcer index was recorded. Ethanol ulcer were produced by 1 mL of ethanol in the rats pretreated with B-Hb 30 min before the ethanol. One hour after ethanol stomach were cut open to score ulcers. Histological examination and analysis of gastric wall mucus, non-protein sulfhydryl groups (NP-SH), and myeloperoxidase (MPO) were carried in gastric tissue following ethanol administration. In control rats pylorus ligation for 6 h resulted in the accumulation of 8.1 ± 0.61 mL of gastric secretion. The treatment of the rats with 100, 300 and 900 mg/kg of B-Hb produced a significant decrease in the volume of gastric secretion 5.6 ± 0.63, 5.5 ± 0.75 and 4.7 ± 0.58 mL respectively as compared to the control group [analysis of variance (ANOVA) F = 4.77, P < 0.05]. The lesion area in the control group was found to be 22.4 ± 3.2 mm(2) six hours after the administration of indomethacin. Treatment of rats with B-Hb at doses of 100 mg/kg (24.3 ± 3.29 mm(2)), 300 mg/kg (16.2 ± 1.45 mm(2)) and 900 mg/kg (12.6 ± 1.85 mm(2)) produced a dose dependent decreased the lesion scores (ANOVA F = 4.50, P < 0.05). The ulcer index following one hour after 1 mL ethanol was 7.1 ± 0.31. Pretreatment of rats with B-Hb at the doses of 100 mg/kg (2.5 ± 0.42), 300 mg/kg (2.1 ± 0.4) and 900 mg/kg (0.7 ± 0.21) significantly inhibited the formation of gastric lesions (ANOVA F = 63.26, P < 0.0001). Histological examination of gastric mucosa following ethanol showed significant lesions in the form of gastric pits with detachment of the surface epithelium; vacuolation of epithelial cells and elongation of microvessels. The changes were dose-dependently attenuated by B-Hb. The treatment of rats with ethanol significantly decreased the Alcian blue binding capacity of gastric wall mucus (480 ± 25.6 μg Alcian blue/g of tissue) as compared to control rats (667 ± 25.8 μg). Pretreatment of rats with B-Hb at the doses of 100 mg/kg (516 ± 31.6 μg/g), 300 mg/kg (558 ± 28.8 μg/g) and 900 mg/kg (654 ± 33.8 μg/g) significantly attenuated ethanol induced depletion of gastric wall mucus (ANOVA F = 8.05, P < 0.005). A significant and dose dependent increase of gastric mucosal NP-SH (ANOVA F = 19.62, P < 0.001) and decrease in MPO activity (ANOVA F = 3.1, P < 0.05) was observed in B-Hb treated rats. B-Hb possesses significant gastric antisecretory and gastroprotective activity against experimentally induced gastric lesion. The gastroprotective effects of B-Hb are accompanied by inhibition of neutrophils activity, reduction of oxidative stress and maintenance of mucosal integrity.

BMP-2 and TGF-β3 do not prevent spontaneous degeneration in rabbit disc explants but induce ossification of the annulus fibrosus

Different approaches for disc regeneration are currently under investigation. Beside gene therapy and tissue engineering techniques, the application of growth and differentiation factors own promising potential. Studies using reduced intervertebral disc models, such as cell or tissue fragment cultures, have limited validity and show controversial results depending on the employed experimental model. Therefore, the goal of the current study was to investigate the effect of BMP-2 and TGF-β3 on intervertebral disc degeneration using an in vitro full-organ disc/endplate culture system. Intervertebral rabbit disc explants were cultured in the presence of 1 μg/ml BMP-2 or TGF-β3 for 21 days in DMEM/F12 media. Nucleus and annulus were analyzed for gene expression of collagen type I and II (Col I/II), aggrecan, collagenases (MMP-1/MMP-13) with RT-qPCR, histological changes with bone and proteoglycan-specific staining (von Kossa, toluidine blue) and differences in cellularity (DNA) and proteoglycan content (alcian blue binding assay). The results demonstrate that disc proteoglycan concentration decreased with time in the TGF-β3 and BMP-2 groups. In the annulus fibrosus (AF), TGF-β3 and BMP-2 resulted in an up-regulation of Col I and type II, and of aggrecan gene expression. In contrast, MMP genes were inhibited. In the nucleus, the growth factors decreased gene expression of aggrecan and spontaneous Col I up-regulation was inhibited by TGF-β3, whereas expression of Col II was decreased with BMP-2. There was no effect on expression of MMP-1 and MMP-13 for most sampling points. However, TGF-β3 and BMP-2 induced ossification of the AF was demonstrated by histology. It can be concluded that both growth factors, at the tested concentrations, may not be suitable to regenerate the whole intervertebral disc organ but they are interesting candidates for being injected alone or in combination into a painful intervertebral disc to induce osseous fusion (spondylodesis).

Rebamipide Increases Mucin-Like Substance Contents and Periodic Acid Schiff Reagent-Positive Cells Density in Normal Rabbits

The effects of rebamipide on the number of periodic acid Schiff reagent (PAS)-positive cells in the conjunctiva, the mucin content in the cornea and conjunctiva of normal rabbits, and desiccation-induced corneal damage in vivo were examined. Rebamipide (0.1%-3%) was applied 6 times a day for 14 days, and the PAS-positive cell count in the bulbar conjunctiva was measured by impression cytology. The amount of conjunctival and corneal mucin-like substances was measured by Alcian blue binding. The corneal damage model was created by desiccation from air flow at room temperature. The level of corneal damage was determined by scoring the area stained with rose bengal and fluorescein dye. Rebamipide increased the number of PAS-positive cells in the conjunctiva when instilled at concentrations of 0.3% or higher, and 1% rebamipide increased the amount of mucin-like substances of the conjunctiva and cornea. Moreover, 1% rebamipide was also found to lower the rose bengal scores of the cornea in the corneal damage model by desiccation. Rebamipide is a possible candidate drug for treatment of cornea and conjunctival epithelial damage due to its mucin-like substance increasing action, for instance, in the treatment of dry eye disease.

Essential oil of Croton zehntneri and its major constituent anethole display gastroprotective effect by increasing the surface mucous layer

Croton zehntneri, a plant native to northeastern Brazil, is widely used in folk medicine to treat gastrointestinal problems and has rich essential oil content. The effects of the essential oil of Croton zehntneri (EOCZ) and its main constituent anethole on several models of gastric lesions were studied in mice and rats. Oral treatment with EOCZ and anethole, both at doses of 30-300 mg/kg, caused similar and dose-dependent gastroprotection against ethanol- and indomethacin-induced gastric damage, but did not change cold-restraint stress-induced ulcers in rats. Furthermore, EOCZ and anethole (both at 30 and 300 mg/kg) similarly and significantly increased the mucus production by the gastric mucosa, measured by Alcian blue binding, in ethanol-induced ulcer model. However, at the same doses, neither EOCZ nor anethole promoted significant alteration in gastric production of non-protein sulfhydryl groups. In pylorus-ligated model, neither EOCZ nor anethole (both at 30 and 300 mg/kg) had a significant effect on the volume of gastric juice, pH, or total acidity. The results of this study show for the first time that EOCZ possesses a gastroprotective potential, an effect mostly attributed to the action of anethole. This activity is related predominantly to the ability of EOCZ and anethole to enhance the production of gastric wall mucus, an important gastroprotective factor. Furthermore, they suggest that EOCZ has potential therapeutic application for the treatment of gastric ulcers.

The Candida albicans histidine kinase Chk1p: Signaling and cell wall mannan

Several published functions associated with the CHK1 histidine kinase of Candida albicans resemble those of the MAPK Cek1p and its cognate receptor Sho1p ( SSU81). To explore this further, we have compared mutants lacking the proteins mentioned above and have constructed a double sho1/chk1Δ null mutant to determine relationships among these proteins. We observed that the sensitivity to Congo red (CR), calcofluor white (CW), as well as clumping of cells, was slightly increased in the double mutant compared to the single chk1Δ or sho1Δ mutants. However, Cek1p phosphorylation via Sho1p, which occurs during log phase growth in the presence or absence of CR in Wt cells, does not require Chk1p. These data suggest that Chk1p and Sho1p are components of parallel but independent signal pathways. In addition, bulk mannan of strains was analyzed by GLC/MS and GPC MALLS and NMR. Compared to Wt and a CHK1 gene-reconstituted strain (CHK23) that contained high, intermediate and low Mw mannan species, we found that the mannan of strains CHK21 ( chk1Δ null), the cek1Δ null, and the double mutant consisted only of low Mw mannan. The sho1Δ null mutant only demonstrated a reduced intermediate type of mannan. Alcian blue binding was lower in cek1Δ, chk1Δ, and the double sho1/chk1Δ null mutant lacking high and intermediate Mw mannan than in the sho1Δ null which had a partial loss of intermediate Mw mannan only. We conclude that the Chk1p HK is part of a functionally similar but parallel pathway to the Sho1p-Cek1p pathway that confers resistance to the cell wall inhibitors CR and CW. However, a functional relationship in mannan biosynthesis of Chk1p and Cek1p exists that only partially requires Sho1p.

INCREASED LEVELS OF GLYCOSAMINOGLYCANS DURING SEPTIC SHOCK

Glycosaminoglycans (GAGs) are structurally heterogeneous negatively charged polysaccharides. Endothelial GAGs, also known as glycocalyx, are involved in capillary permeability. In rat venules stimulated with proinflammatory substances ex vivo, the GAG-containing proteoglycan, syndecan-1, is shed from the endothelium. We wanted to investigate if we could trace the same response during septic shock as reflected in the circulating GAG levels. Arterial plasma samples were collected from 18 consecutive septic shock patients admitted to our intensive care unit. Plasma GAGs were measured with an Alcian blue slot binding assay, and syndecan-1 levels were measured with enzyme-linked immunosorbent assay. Effects of GAGs on the antibacterial activity of plasma were assessed by a radial diffusion assay. The median plasma GAG level was significantly higher in the septic shock patients than in matched controls (median [interquartile range], 2.7 microg/mL [1.9 - 4.8 microg/mL] vs. 1.8 microg/mL [1.7 - 2.0 microg/mL]). Furthermore, the GAG levels were significantly higher in nonsurvivors (4.6 microg/mL [3.1 - 8.8 microg/mL], n = 8) than survivors (1.8 microg/mL [1.6 - 2.6 microg/mL], n = 10). The syndecan-1 levels were also increased in the patients compared with controls (246 ng/mL [180 - 496 ng/mL] vs. 26 ng/mL [23 - 31 ng/mL]) and correlated to the cardiovascular Sequential Organ Failure Assessment (SOFA) score. The GAGs inhibited the endogenous antibacterial activity of plasma as well as isolated antimicrobial peptides. The concentrations required were in the same range as the GAG levels measured in the patients. These results show that the GAG levels are increased in septic shock patients, possibly reflecting peripheral endothelial cell damage. We also found that GAGs in relevant concentrations neutralize antimicrobial peptides in plasma.

Effect of acute bilateral adrenalectomy and reserpine on gastric mucus secretion and mucosal injury in pyloric ligated rats

Adrenalectomy produces many changes in gastric functional parameters including gastric acid secretion, gastric motility and mucosal blood flow. The present study was undertaken to assess the effect of acute bilateral adrenalectomy and reserpine on gastric mucus secretion and degradation in the pyloric ligated rats. Groups of Wistar rats of mixed sexes were adrenalectomized or administered with 5 mg/kg reserpine intraperitoneally or given both treatment followed by pyloric ligation. Adherent mucus was assessed by the alcian blue binding technique while erosion of the surface gel was assessed from the appearance of sialic acids and galactose in the gastric juice. Gastric mucosa injury was quantified by measuring the area of gastric mucosa damage. The amount of alcian blue bound to adherent mucus and soluble mucin concentration were not significantly affected by adrenalectomy (P > 0.05). Reserpine diminished adherent mucus secretion and increased soluble glycoprotein in the gastric juice. Adrenalectomized animals (40%, n = 5) developed haemorrhagic lesions as compared to 100% (n = 5) in animals treated with reserpine. Adrenalectomy or propranolol did not protect the animals against the reserpine induced injury. It is concluded that the adrenal glands seem to be of no importance in the secretion of gastric mucus but reserpine inhibits mucus release and stimulates surface mucus gel breakdown via β-adrenoceptor stimulation.

Ulcer preventive and antioxidative properties of astaxanthin from Haematococcus pluvialis

The anti-ulcer properties of astaxanthin fractions such as total carotenoid and astaxanthin esters from Haematococcus pluvialis were evaluated in ethanol-induced gastric ulcers in rats. Since oxygen radical release is a pathogenic factor of ethanol-induced gastric damage, astaxanthin — a free radical scavenger, was investigated as a potential ulcer preventive agent. Astaxanthin fractions — total carotenoid and astaxanthin esters were orally administered to experimental rats at 100, 250 and 500 μg/kg b.w. prior to ulcer induction. Alcian blue binding assay indicates that, total carotenoid and astaxanthin esters at 500 μg/kg b.w could protect gastric mucin ~ 40% and 67% respectively. Pre-treatment with astaxanthin esters, also resulted in significant increase in antioxidant enzyme levels — catalase, superoxide dismutase, and glutathione peroxidase in stomach homogenate. Histopathological examination substantiated the protective effect of astaxanthin in pre-treated rats. The increased antioxidant potencies such as free radical scavenging activity with an IC 50 of ~ 8 μg/ml and reducing power abilities (59 × 10 3 U/g) in vitro, reveal that H. pluvialis astaxanthin may protect gastric mucosal injury by antioxidative mechanism. In addition, ~ 23 fold increased lipoxygenase-inhibitory property, in comparison with standard astaxanthin and significant H +, K +-ATPase-inhibitory activity of astaxanthin esters, in comparison with known proton pump blocking anti-ulcer drug — omeprazole, may envisage the potential gastroprotective effect by regulating the gastric mucosal injury and gastric acid secretion by the gastric cell during ulcer disease.

Decreased lubricin concentrations and markers of joint inflammation in the synovial fluid of patients with anterior cruciate ligament injury.

To study the effect of anterior cruciate ligament (ACL) injury on lubricin concentrations in synovial fluid (SF) and its correlation with time postinjury, inflammatory cytokines, lubricin-degrading enzymes, and SF proteoglycan content. SF samples were obtained from both knees of 30 patients with unilateral ACL insufficiency, 32-364 days postinjury. Lubricin, inflammatory cytokines (interleukin-1beta [IL-1beta], tumor necrosis factor alpha [TNFalpha], and IL-6), and catabolic enzymes (procathepsin B and neutrophil elastase) were measured in SF from injured and contralateral (uninjured) joints, by enzyme-linked immunosorbent assay. Sulfated glycosaminoglycan (sGAG) levels in the SF were measured by Alcian blue binding assay. SF lubricin concentrations were significantly (P < 0.001) reduced at an early stage following ACL injury when compared with those in the contralateral joint. Within 12 months, the lubricin concentration in the injured knee (slope = 0.006, SE = 0.00010, P < 0.001) approached that in the contralateral knee, which did not change with time (slope = -0.0002, SE = 0.00050, P = 0.71). TNFalpha levels showed a significant negative relationship with log2 lubricin levels. IL-1beta, TNFalpha, IL-6, procathepsin B, and neutrophil elastase concentrations in SF from injured knees were greater in samples from recently injured knees compared with those that were chronically injured. There were no detectable cytokines or enzymes in the SF of contralateral joints. Concentrations of sGAG were significantly (P = 0.0002) higher in the SF from injured knees compared with the contralateral joints. The decrease in SF lubricin concentrations following ACL injury may place the joint at an increased risk of wear-induced damage as a consequence of lack of boundary lubrication, potentially leading to secondary osteoarthritis. The decrease in SF lubricin was associated with an increase in levels of inflammatory cytokines.

A Phenomics Approach in Yeast Links Proton and Calcium Pump Function in the Golgi

The Golgi-localized Ca2+- and Mn2+-transporting ATPase Pmr1 is important for secretory pathway functions. Yeast mutants lacking Pmr1 show growth sensitivity to multiple drugs (amiodarone, wortmannin, sulfometuron methyl, and tunicamycin) and ions (Mn2+ and Ca2+). To find components that function within the same or parallel cellular pathways as Pmr1, we identified genes that shared multiple pmr1 phenotypes. These genes were enriched in functional categories of cellular transport and interaction with cellular environment, and predominantly localize to the endomembrane system. The vacuolar-type H+-transporting ATPase (V-ATPase), rather than other Ca2+ transporters, was found to most closely phenocopy pmr1Delta, including a shared sensitivity to Zn2+ and calcofluor white. However, we show that pmr1Delta mutants maintain normal vacuolar and prevacuolar pH and that the two transporters do not directly influence each other's activity. Together with a synthetic fitness defect of pmr1DeltavmaDelta double mutants, this suggests that Pmr1 and V-ATPase work in parallel toward a common function. Overlaying data sets of growth sensitivities with functional screens (carboxypeptidase secretion and Alcian Blue binding) revealed a common set of genes relating to Golgi function. We conclude that overlapping phenotypes with Pmr1 reveal Golgi-localized functions of the V-ATPase and emphasize the importance of calcium and proton transport in secretory/prevacuolar traffic.