Normal Hamsters Research Articles

Effect of Berberine on promoting the excretion of cholesterol in high-fat diet-induced hyperlipidemic hamsters.

BackgroundBerberine (BBR), as a new medicine for hyperlipidemia, can reduce the blood lipids in patients. Mechanistic studies have shown that BBR activates the extracellular-signal regulated kinase pathway by stabilizing low-density-lipoprotein receptor mRNA. However, aside from inhibiting the intestinal absorption of cholesterol, the effects of BBR on other metabolic pathways of cholesterol have not been reported. This study aimed to investigate the action of BBR on the excretion of cholesterol in high-fat diet-induced hyperlipidemic hamsters.MethodsGolden hamsters were fed a high-fat diet (HFD) for 6 weeks to induce hyperlipidemia, followed by oral treatment with 50 and 100 mg/kg/day of BBR or 10 and 30 mg/kg/day of lovastatin for 10 days, respectively. The levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), transaminases, and total bile acid in the serum, liver, bile and feces were measured using an enzyme-linked immunosorbent assay. The cholesterol (as well as coprostanol) levels in the liver, bile and feces were determined by gas chromatography–mass spectrometry.ResultsThe HFD hamsters showed significantly hyperlipidemic characteristics compared with the normal hamsters. Treatment with BBR for 10 days reduced the serum TC, TG and LDL-C levels in HFD hamsters by 44–70, 34–51 and 47–71 %, respectively, and this effect was both dose- and time-dependent. Initially, a large amount of cholesterol accumulated in the hyperlipidemic hamster livers. After BBR treatment, reductions in the liver cholesterol were observed by day 3 and became significant by day 7 at both doses (P < 0.001). Meanwhile, bile cholesterol was elevated by day 3 and significantly increased at day 10 (P < 0.001). BBR promoted cholesterol excretion from the liver into the bile in hyperlipidemic hamsters but not in normal hamsters, and these results provide a link between the cholesterol-lowering effect of BBR with cholesterol excretion into the bile.ConclusionsWe conclude that BBR significantly promoted the excretion of cholesterol from the liver to the bile in hyperlipidemic hamsters, which led to large decreases in the serum TC, TG and LDL-C levels. Additionally, compared with lovastatin, the BBR treatment produced no obvious side effects on the liver function.

Identification, synthesis, and safety assessment of forchlorfenuron (1-(2-chloro-4-pyridyl)-3-phenylurea) and its metabolites in kiwifruits.

Identification and evaluation of safety of forchlorfenuron ((1-(2-chloro-4-pyridyl)-3-phenylurea)), 1, metabolites after biotransformation in kiwifruit is the objective of this study. To elucidate properties of these metabolites, liquid chromatography hybrid ion trap time-of-flight mass spectrometry (LC-IT-TOF-MS) was applied, with MetID Solution and Formula Predictor Software in positive mode. Cytotoxicity of forchlorfenuron and its metabolites were tested through sulforhodamine B assays against normal Chinese hamster ovary cells (CHO). As deduced from characteristic fragment ions of forchlorfenuron, then confirmed by comparison with synthetic standards, as well as characterized by NMR and mass spectrometry techniques, results indicate the presence of 4-hydroxyphenyl-forchlorfenuron, 2, 3-hydroxyphenyl-forchlorfenuron, 3, and forchlorfenuron-4-O-β-D-glucoside, 5. Forchlorfenuron (IC50 = 12.12 ± 2.14 μM) and 4-hydroxyphenyl-forchlorfenuron (IC50 = 36.15 ± 1.59 μM), exhibits significant cytotoxicity against CHO, while 3-hydroxyphenyl-forchlorfenuron and forchlorfenuron-4-O-β-D-glucoside show no cytotoxicity.

Cytotoxicity screening of essential oils in cancer cell lines

This study evaluated the cytotoxicity activity of the essential oils of Tagetes erecta L., Asteraceae (TE-OE), Tetradenia riparia (Hochst.) Codd, Lamiaceae (TR-OE), Bidens sulphurea (Cav.) Sch. Bip., Asteraceae (BS-OE), and Foeniculum vulgare Mill., Apiaceae (FV-OE), traditionally used in folk medicine, against the tumor cell lines murine melanoma (B16F10), human colon carcinoma (HT29), human breast adenocarcinoma (MCF-7), human cervical adenocarcinoma (HeLa), human hepatocellular liver carcinoma (HepG2), and human glioblastoma (MO59J, U343, and U251). Normal hamster lung fibroblasts (V79 cells) were included as control. The cells were treated with essential oil concentrations ranging from 3.12 to 400μg/ml for 24h. The cytotoxic activity was evaluated using the XTT assay; results were expressed as IC50, and the selectivity index was calculated. The results were compared with those achieved for classic chemotherapeutic agents. TE-OE was the most promising among the evaluated oils: it afforded the lowest IC50 values for B16F10 cells (7.47±1.08μg/ml) and HT29 cells (6.93±0.77μg/ml), as well as selectivity indices of 2.61 and 2.81, respectively. The major BS-EO, FV-EO and TE-EO chemical constituents were identified by gas chromatography mass spectrometry as being (E)-caryophyllene (10.5%), germacrene D (35.0%) and 2,6-di-tert-butyl-4-methylphenol (43.0%) (BS-EO); limonene (21.3%) and (E)-anethole (70.2%) (FV-EO); limonene (10.4%), dihydrotagetone (11.8%), α-terpinolene (18.1%) and (E)-ocimenone (13.0%) (TE-EO); and fenchone (6.1%), dronabinol (11.0%), aromadendrene oxide (14.7%) and (E,E)–farnesol (15.0%) (TR-EO). 2,6-di-tert-butyl-4-methylphenol (43.0%), (E)-anethole (70.2%) and α-terpinolene (18.1%), respectively. These results suggest that TE-OE may be used to treat cancer without affecting normal cells.

Serum perfluorooctanoic acid (PFOA) concentrations in normal and hyperlipidemic female hamsters dosed orally with ammonium perfluorooctanoate (APFO) for up to 30 days

In epidemiology studies, the presence of perfluorooctanoate (PFOA) in human blood has been associated with higher serum cholesterol concentrations. A possible explanation for these results is that elevated serum cholesterol might reduce clearance of PFOA. In this study, female hamsters, which transport and regulate cholesterol in a manner similar to humans, were fed normal diet or diet supplemented with 0.05% cholesterol and 10% coconut oil (high-fat diet) resulting in hyperlipidemia throughout the study in supplemented animals. Hamsters on either a normal and high-fat diet were given oral doses of 0.1, 1.0, or 10mg APFO/kg for 30 days. Serum PFOA concentrations evaluated 24h after 1, 10, 20, and 30 doses of APFO were not altered in hyperlipidemic hamsters compared to those fed normal diet. For a given dose group, serum concentrations of PFOA were highest following the 10 doses (except for the 10mg/kg group where concentrations were the highest after the first dose) and were lowest after 20 and 30 doses. Under the condition of this study, higher serum lipids did not affect the absorption and clearance of serum PFOA. Serum PFOA concentrations declined over the course of the study despite continued daily dosing with APFO. This does not support the hypothesis that higher serum lipids might increase the retention of PFOA in the body.

Asperaculanes A and B, two sesquiterpenoids from the fungus Aspergillus aculeatus.

Six sesquiterpenoids 1–6, including two new ones, an ent-daucane-type sesquiterpenoid, asperaculane A (1), and a nordaucane one, asperaculane B (2), and four known nordaucane derivatives, aculenes A–D 3–6, together with the known secalonic acid D (7), were isolated from a fermentation culture of the fungus Aspergillus aculeatus. Their structures and absolute configurations were established by analyses of their spectroscopic data, including 1D and 2D-NMR spectra, HR-ESIMS, electronic circular dichroism (ECD) data, and quantum chemical calculations. These metabolites were evaluated for in vitro cytotoxic activity against two cell lines, human cancer cell lines (HeLa) and one normal hamster cell line (CHO).

Functionalization of luminescent YVO4:Eu3+ nanoparticles by sol–gel

Over the last decades, researchers have explored nanotechnological applications in different areas. The non-hydrolytic and hydrolytic sol–gel routes offer the ideal conditions to obtain materials with distinct compositions and multifunctionality, for use in such diverse areas as nanomedicine and technology. In this work, we used the modified hydrolytic sol–gel route to prepare YVO4 doped with Eu3+ ion. The YVO4:Eu3+ nanoparticles were functionalized with 3-chloropropyltriethoxysilane using the hydrolytic sol–gel process; the drug cisplatin was then added to them. The final powder was characterized by thermal analysis, infrared spectroscopy, X-ray diffraction, and photoluminescence. The powder X-ray diffraction patterns of the samples obtained before and after functionalization revealed well defined peaks ascribed to the tetragonal structure of the YVO4 phase. The thermal analysis curves evidenced mass loss relative to 3-chloropropyltriethoxysilane and cisplatin decomposition. Infrared spectroscopy showed the peaks related to the CH and NH groups vibration modes, confirming YVO4 functionalization. The excitation and emission spectrum of the Eu3+ ion did not change upon its doping into the matrix functionalized with 3-chloropropyl and cisplatin. Cytotoxicity tests conducted on normal Chinese hamster (V79 cells) and murine melanoma (B16F10) cells attested that the matrix was not toxic.

Hydrogen gas attenuates embryonic gene expression and prevents left ventricular remodeling induced by intermittent hypoxia in cardiomyopathic hamsters.

The prevalence of sleep apnea is very high in patients with heart failure (HF). The aims of this study were to investigate the influence of intermittent hypoxia (IH) on the failing heart and to evaluate the antioxidant effect of hydrogen gas. Normal male Syrian hamsters (n = 22) and cardiomyopathic (CM) hamsters (n = 33) were exposed to IH (repeated cycles of 1.5 min of 5% oxygen and 5 min of 21% oxygen for 8 h during the daytime) or normoxia for 14 days. Hydrogen gas (3.05 vol/100 vol) was inhaled by some CM hamsters during hypoxia. IH increased the ratio of early diastolic mitral inflow velocity to mitral annulus velocity (E/e', 21.8 vs. 16.9) but did not affect the LV ejection fraction (EF) in normal Syrian hamsters. However, IH increased E/e' (29.4 vs. 21.5) and significantly decreased the EF (37.2 vs. 47.2%) in CM hamsters. IH also increased the cardiomyocyte cross-sectional area (672 vs. 443 μm(2)) and interstitial fibrosis (29.9 vs. 9.6%), along with elevation of oxidative stress and superoxide production in the left ventricular (LV) myocardium. Furthermore, IH significantly increased the expression of brain natriuretic peptide, β-myosin heavy chain, c-fos, and c-jun mRNA in CM hamsters. Hydrogen gas inhalation significantly decreased both oxidative stress and embryonic gene expression, thus preserving cardiac function in CM hamsters. In conclusion, IH accelerated LV remodeling in CM hamsters, at least partly by increasing oxidative stress in the failing heart. These findings might explain the poor prognosis of patients with HF and sleep apnea.

Effects of short‐term oral administration of trimethoprim‐sulfamethoxazole on tear production in clinically normal Syrian hamsters

To determine effects of 14 days of oral administration of trimethoprim-sulfamethoxazole on aqueous tear production in clinically normal Syrian hamsters. Fifteen healthy adult Syrian hamsters. Baseline phenol red thread test (PRTT) values were recorded 1 day prior to treatment. Eight hamsters in treated group received 15 mg/kg trimethoprim-sulfamethoxazole orally twice a day for 14 days. The remaining seven hamsters were used as untreated controls and received a placebo. All ophthalmic tests were performed without chemical restraint. PRTT values were evaluated in both eyes of all Syrian hamsters using a commercial PRTT strip of a single lot number. The mean ± SD baseline PRTT values for treatment group and control group were 7.25 ± 1.3 mm/15 s and 5.42 ± 1.13 mm/15 s, respectively. After 14 days of drug administration, the mean ± SD PRTT values for treatment group and control group were 5.62 ± 1.5 and 5.64 ± 0.74, respectively. Significant decreases in the PRTT values, compared with baseline values, were observed in the treatment group on day 15 (P = 0.042). Trimethoprim-sulfamethoxazole decreased tear production in the hamster in this study.

Islet neogenesis-associated protein (INGAP): The role of its endogenous production as a positive modulator of insulin secretion

Islet neogenesis-associated protein (INGAP) is a peptide found in pancreatic exocrine-, duct- and islet- non-β-cells from normal hamsters. Its increase induced by either its exogenous administration or by the overexpression of its gene enhances β-cell secretory function and increases β-cell mass by a combination of stimulation of cell replication and islet neogenesis and reduction of β-cell apoptosis. We studied the potential modulatory role of endogenous INGAP in insulin secretion using two different experimental approaches. Hamster islets transfected with INGAP-small interfering RNA (INGAP-siRNA) were used to study glucose-stimulated insulin secretion (GSIS). In parallel, freshly isolated islets were incubated with high glucose and the same concentration of either a specific anti-INGAP rabbit serum or normal rabbit serum. INGAP-siRNA transfected islets reduced their INGAP mRNA and protein content by 35.1% and 47.2%, respectively whereas GSIS decreased by 25.8%. GSIS by transfected islets attained levels comparable to those recorded in control islets when INGAP pentadecapeptide (INGAP-PP) was added to the culture medium. INGAP antibody in the medium decreased significantly GSIS in a dose-dependent manner. These results indicate that endogenous INGAP plays a “physiological” positive modulatory role in insulin secretion, supporting its possible use in the treatment of prediabetes and Type 2 diabetes.

Semisynthesis and in vitro cytotoxic evaluation of new analogues of 1-O-acetylbritannilactone, a sesquiterpene from Inula britannica

Semisynthetic analogues of the natural product 1-O-acetylbritannilactone (ABL), a sesquiterpene isolated from the medicinal plant Inula britannica, have been prepared and exhibited significant in vitro cytotoxic activities against four cell lines including three human cancer cell lines (HCT116, HEp-2 and HeLa) and one normal hamster cell line (CHO). Structure–activity relationships indicate that esterification of 6-OH (enhanced lipophilicity) and α-methylene-γ-lactone functionalities play important roles in conferring cytotoxicity. Among the tested compounds, 14 bearing a lauroyl group (12C) at the 6-OH position displayed most potent in vitro cytotoxic activity, with IC50 values between 2.91 and 6.78 μM, comparable to the positive control etoposide (VP-16, IC50 values between 2.13 and 4.79 μM). Moreover, the compound 14 triggered remarkable apoptosis at a low concentration, and induced cell cycle arrest in G2/M phase in HCT116 cells. The biological assays conducted with normal cells (CHO) revealed that all the synthetic compounds are no selective against cancer cell lines tested.

Interaction between 14-3-3β and PrP influences the dimerization of 14-3-3 and fibrillization of PrP106–126

Proteins of the 14-3-3 family are universal participate in multiple cellular processes. However, their exact role in the pathogenesis of prion diseases remains unclear. In this study, we proposed that human PrP was able to form molecular complex with 14-3-3β. The domains responsible for the interactions between PrP and 14-3-3β were mapped at the segments of amino acid (aa) residues 106–126 within PrP and aa 1–38 within 14-3-3β. Homology modeling revealed that the key aa residues for molecular interaction were D22 and D23 in 14-3-3β as well as K110 in PrP. Mutations in these aa residues inhibited the interaction between the two proteins in vitro. Our results also showed that recombinant PrP encouraged 14-3-3β dimer formation, whereas PrP106–126 peptide inhibited it. Recombinant 14-3-3β disaggregated the mature PrP106–126 fibrils in vitro. Moreover, the PrP–14-3-3 protein complexes were observed in the brain tissues of normal and scrapie agent 263K infected hamsters. Colocalization of PrP and 14-3-3 was seen in the cytoplasm of human neuroblastoma cell line SH-SY5Y, as well as human cervical cancer cell line HeLa transiently expressing full-length human PrP. Our current data suggest the neuroprotection of PrPC and neuron damage caused by PrPSc may be associated with their functions of 14-3-3 dimerization regulation.

MRI and 1H MRS evaluation for the serial bile duct changes in hamsters after infection with Opisthorchis viverrini

A 3T MR scanner was used to investigate the relationship between the alteration of bile duct lesions and the hepatic metabolic changes in hamsters infected with Opisthorchis viverrini by using 3T MRI and 1H MR spectroscopy. Animals were divided into control and infected groups. Five normal hamsters were used as control; fifty-five hamsters were infected with O. viverrini to induce bile duct lesions and hepatic metabolic changes. T2-weighted image sequence in three orthogonal planes were conducted by MRI scans. Single-voxel 1H MRS was performed to obtain the relative choline-to-lipid ratios. The livers and bile ducts were excised for the histologic examination. The progression of bile duct changes by histology and metabolic changes in O. viverrini infected hamsters were co-investigated. In the O. viverrini-infected group, the T2-weighted images revealed the time-dependent intra- and extra-hepatic duct dilatations in the liver. The mean (±SD) choline-to-lipid ratios were 0.11±0.035 in the control group, whereas the ratio in the infected group increased significantly with the progression of time. Histologic grading of hepatic inflammation and fibrosis were correlated well with the MRI grading (Spearman rank correlation test; r=0.746 and p<0.001). The control group showed no dilatation of the bile ducts and showed normal liver patterns. Noninvasive technique, MRI and 1H MRS can demonstrated and applied to evaluate not only the inflammation-related fibrosis in the small bile ducts but also the metabolic changes in the liver induced by O. viverrini infection. A significant increase in the choline-to-lipids ratios were observed in parallel with the time-course of infection. O. viverrini infected in human is detected by stool examination. Hepatobiliary morbidity is detected and followed up by ultrasonography. MRI and MRS can be used in conjunction with ultrasonography for evaluation of progression of the disease.

Cyanines in photodynamic reaction assisted by reversible electroporation—in vitro study on human breast carcinoma cells

Ineffective drug delivery is a vast problem of anticancer therapies. The aim of this study was to investigate the possibility of enhancement of cyanines transport through the cell membrane by electroporation and to evaluate a photodynamic activity of these compounds. We evaluated in vitro the effectiveness of photodynamic reaction with cyanines on breast adenocarcinoma cells (MCF-7/WT) and normal Chinese hamster ovary cells (CHO) lacking voltage-dependent ion channels, alone and combined with electropermeabilization. Among six cyanines tested, two compounds could be indicated as good therapeutic candidates: IR-775 and IR-786. Cellular effects were assessed with MTT assay reporting cell mitochondrial activity and with SRB assay based on the measurement of cellular protein content. Cyanines localization was observed with confocal microscope. Photodynamic reaction of MCF-7/WT cells with IR-775 and IR-786 did not result in cellular dysfunction. Electric field intensities and pulse duration, non-toxic for cells, significantly increased photocytotoxicity of the cyanines after electropermeabilization with IR-775 and IR-786. Much shorter exposure times were efficient for cyanines in photodynamic reaction assisted by electroporation (10 min instead of 24h). Our results indicate that electroporation of cancerous cells in the presence of cyanine dyes could increase the uptake of the photosensitizer, which correlates with a higher cytotoxicity in the breast adenocarcinoma cell line. Electroporation may be an attractive delivery system for photosensitizers in photodynamic therapy, enabling application of new compounds and reduction of drug dose and exposure time.

Tumor-Related Gene Changes in Immunosuppressive Syrian Hamster Cholangiocarcinoma

The results of a previous study demonstrated that prednisolone enhanced cholangiocarcinogenesis. Therefore, to clarify molecular changes during immunosuppressive cholangiocarcinogenesis, Syrian hamsters were divided into 8 groups: uninfected controls; immunosuppressed Syrian hamsters using prednisolone (P); normal Syrian hamsters administered N-nitrosodimethylamine (ND); immunosuppressed Syrian hamsters administered N-nitrosodimethylamine (NDis); normal Syrian hamsters infected with Opisthorchis viverrini (OV); immunosuppressed Syrian hamsters infected with O. viverrini (OVis); normal Syrian hamsters infected with O. viverrini and administered N-nitrosodimethylamine (CCA); and immunosuppressed Syrian hamsters infected with O. viverrini and administered N-nitrosodimethylamine (CCAis). Syrian hamster livers were used for analysis of tumor-related gene expression and immunohistochemistry through cytokeratin 19 (CK19) and proliferating cell nuclear antigen (PCNA) staining. The tumor-related gene expression results show that CCAis groups at all time points exhibited upregulation of COX-2, IL-6, SOD1, CAT and iNOS and downregulation of p53, which correlated with the predominant expression of CK19 and PCNA in liver tissue. These results suggest that prednisolone enhances cholangiocarcinoma development, which was confirmed by molecular changes.

Diagnosis of Fasciola gigantica infection using a monoclonal antibody-based sandwich ELISA for detection of circulating cathepsin B3 protease

A reliable monoclonal antibody (MoAb)-based sandwich enzyme-linked immunosorbent assay (sandwich ELISA) was developed for the detection of circulating cathepsin B3 protease (CatB3) in the sera from mice experimentally infected with Fasciola gigantica and cattle naturally infected with the same parasite. The MoAb 2F9 and biotinylated rabbit polyclonal anti-recombinant CatB3 antibody were selected due to their high reactivities and specificities to F. gigantica CatB3 antigen based on indirect ELISA and immunoblotting. The lower detection limit of the sandwich ELISA assay was 10, 100 and 400pg/ml, when applied for the detection of rCatB3 antigen and CatB3 in whole body (WB) of newly excysted juveniles (NEJ) and metacercariae (Met) of F. gigantica, respectively. This sandwich ELISA assay could detect F. gigantica infection from day 1 to 35 post infection and revealed that circulating level of CatB3 peaked at day 1 post infection. In contrast, the antibody detection by indirect ELISA could only demonstrate the antibody level from 35 days post infection. The reliability of the assay method was evaluated using serum samples from mice infected with F. gigantica or Schistosoma mansoni, and hamsters infected with Opisthorchis viverrini, as well as normal mice and hamsters. In addition, sera from cattle infected with Paramphistomum cervi, Strongylid, Trichuris sp. and Strongyloides sp., as well as sera from normal cattle were also assessed. In experimental mice, the diagnostic sensitivity, specificity, positive predictive value, negative predictive value, false positive rate, false negative rate and accuracy of ELISA were 95%, 100%, 100%, 97.9%, 0%, 5.3% and 98.5%, while in natural cattle they were 96.7%, 100%, 100%, 98.5%, 0%, 3.4% and 98.9%, respectively. Hence, this assay method showed high efficient and precision for early diagnosis of fasciolosis by F. gigantica.

Electric field-assisted delivery of photofrin to human breast carcinoma cells.

The influence of electroporation on the Photofrin uptake and distribution was evaluated in the breast adenocarcinoma cells (MCF-7) and normal Chinese hamster ovary cells (CHO) lacking voltage-dependent channels in vitro. Photofrin was used at a concentration of 5 and 25 μM. The uptake of Photofrin was assessed using flow cytometry and fluorescence microscopy methods. Cells viability was evaluated with crystal violet assay. Our results indicated that electropermeabilization of cells, in the presence of Photofrin, increased the uptake of the photosensitizer. Even at the lowest electric field intensity (700 V/cm) Photofrin transport was enhanced. Flow cytometry results for MCF-7 cells revealed ~1.7 times stronger fluorescence emission intensity for cells exposed to Photofrin and electric field of 700 V/cm than cells treated with Photofrin alone. Photofrin was effective only when irradiated with blue light. Our studies on combination of photodynamic reaction with electroporation suggested improved effectiveness of the treatment and showed intracellular distribution of Photofrin. This approach may be attractive for cancer treatment as enhanced cellular uptake of Photofrin in MCF-7 cells can help to reduce effective dose of the photosensitizer and exposure time in this type of cancer, diminishing side effects of the therapy.

Results of phenol red thread test in clinically normal Syrian hamsters (Mesocricetus auratus)

To determine the normal reference range for phenol red thread test (PRTT) values in clinically normal Syrian hamsters (Mesocricetus auratus). Sixteen healthy adult Syrian hamsters (eight males and eight females) were used in this study. Ophthalmic examinations were performed without chemical restraint. PRTT values were evaluated in both eyes of all Syrian hamsters using a commercial PRTT strip of a single lot number. No statistically significant differences between right and left eyes were found for any of the results. The mean ± SD PRTT values for the study population were 6.8 ± 2.5 mm/15 s with a range from 3 to 11.5 mm/15 s. Mean PRTTs in male animals were 5.1 ± 1.2 mm/15 s, whereas mean PRTTs in female hamsters were 8.5 ± 2.3 mm/15 s. Comparison between mean PRTT values in males and females showed a significant difference (P = 0.004). Mean weights for males and females were 80.9 ± 4.8 and 90.6 ± 8.5 g, respectively. No linear relationship between mean PRTT and body weight was found in female (P = 0.46) and male (P = 0.92) hamsters. This study provides novel data for normal reference ranges of PRTT values in healthy Syrian hamsters. Results of this study may assist veterinarians in the diagnosis of ocular surface disease and syndromes affecting the tear film in these species.

Chymase inhibitor ameliorates hepatic steatosis and fibrosis on established non‐alcoholic steatohepatitis in hamsters fed a methionine‐ and choline‐deficient diet

Chymase plays a role in the augmentation of angiotensin II formation, which is involved in liver fibrosis. The therapeutic effects of a chymase inhibitor, TY-51469, on established hepatic steatosis and fibrosis were investigated in a model of developed non-alcoholic steatohepatitis. Hamsters were fed a normal diet or methionine- and choline-deficient (MCD) diet for 12 weeks. Then, treatment with TY-51469 (1 mg/kg per day) or placebo was initiated, and the treatment was continued concurrently with the MCD diet for an additional 12 weeks. At 12 weeks after initiating the MCD diet, marked hepatic steatosis and fibrosis were observed in MCD diet-fed hamsters. Malondialdehyde and gene expression levels of collagen I, collagen III, α-smooth muscle actin (α-SMA) and Rac-1 in liver extracts were also increased in the MCD-diet-fed hamsters at 12 weeks. At 24 weeks, hepatic steatosis and fibrosis were more prominent in the placebo-treated hamsters that were fed the MCD-diet for 24 weeks versus 12 weeks. Hamsters treated with TY-51469 for 12 weeks after being on a 12-week MCD diet had significant ameliorations in both hepatic steatosis and fibrosis, and there were no significant differences compared to normal diet-fed hamsters. There were significant augmentations in angiotensin II and malondialdehyde, and gene expressions of collagen I, collagen III, α-SMA and Rac-1 in the placebo-treated hamsters at 24 weeks; however, these levels were reduced to normal levels in the TY-51469-treated hamsters. TY-51469 not only prevented the progression of hepatic steatosis and fibrosis, but also ameliorated hepatic steatosis and fibrosis.

PrP octarepeats region determined the interaction with caveolin-1 and phosphorylation of caveolin-1 and Fyn

Caveolin-1 is one of the major constituents of caveolae. Both Cav-1 and PrP are plasma membrane proteins, which show active capacities for molecular interactions with many other proteins or agents, including themselves. Using yeast two-hybrid system and immunoprecipitation, we reconfirmed the molecular interaction between human Cav-1 and PrP. With co-immunoprecipitation tests, PrP(C)-Cav-1 and PrP(Sc)-Cav-1 complexes were identified in the brain homogenates of normal and scrapie agent 263K-infected hamsters, respectively. Transient expression of wild-type PrP (PrP-PG5) in HEK293 cells did not change the situation of Cav-1 and subsequent signal transduction pathways, while cross-linking of the expressed PrP with specific antibody induced remarkable colocalization of PrP and Cav-1 on the plasma membrane and significant increases of phosphorylated Cav-1 and phosphorylated Fyn. With deleted and inserted PrP mutants within octarepeat region, we observed obvious octarepeat-associated phenomena, including lower binding capacity with Cav-1 in vitro, unable to co-localize with Cav-1 in the cells and to induce up-regulation of p-Cav-1 and p-Fyn when removal of octarepeats in the context of full-length PrP. Moreover, we found that treatment on HEK293 cells with fibrous form of recombinant PrP protein led to up-regulating the levels of p-Cav-1 and p-Fyn. Our data here provide strong evidence that octarepeats of PrP are critical for the interaction between PrP and Cav-1. Significant alterations in the cultured cells, either the distributions of PrP and Cav-1 morphologically or the up-regulations of p-Cav-1 and p-Fyn, induced by antibody-mediated cross-linking or fibrous forms of PrP may suggest a possible internalization process of PrP(Sc).

Topical rapamycin suppresses the angiogenesis pathways induced by pulsed dye laser: Molecular mechanisms of inhibition of regeneration and revascularization of photocoagulated cutaneous blood vessels

Pulsed dye laser (PDL) is the most effective treatment for port wine stain (PWS) birthmarks. However, regeneration and revascularization of photocoagulated blood vessels may result in poor therapeutic outcome. We have recently shown that rapamycin (RPM), an angiogenesis inhibitor, can reduce the regeneration and revascularization of photocoagulated blood vessels. Herein, we attempt to further elucidate the molecular pathophysiology on the inhibition of the regeneration and revascularization of photocoagulated blood vessels by topical RPM in an animal model. Two separate skin areas on each hamster were irradiated by PDL. After PDL exposure, topical RPM was applied daily to one of the randomly selected test sites. PDL, PDL + RPM and normal skin test sites were biopsied on day 3 after PDL exposure. The total ribonucleic acid (RNA) and protein were extracted from biopsied skin samples and quantified. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunoblot were subsequently performed to quantify the mRNA and protein levels of hypoxia-inducible factor-1alpha (HIF-1α), vascular endothelial growth factor (VEGF) and ribosomal protein S6 kinase (S6). The phosphorylation levels of S6 and AKT were also evaluated by immunoblot. The mRNA and protein levels of HIF-1α, VEGF, and S6 significantly increased after PDL exposure as compared to the normal hamster skin. Topical application of 1% RPM suppressed the PDL-induced increase in mRNA and protein levels of those genes on day 3 post-PDL exposure. The phosphorylation levels of S6 and AKT increased after PDL exposure but the increases were suppressed by the topical application of RPM. The increase in expression of HIF-1α, VEGF, and S6 after PDL-exposure suggests that angiogenesis pathways play very active roles in the process of skin blood vessel regeneration and revascularization. Topical application of 1% RPM can suppress the angiogenesis pathways and, therefore, reduce the regeneration and revascularization of photocoagulated blood vessels.