In vitro toxicity of melamine against Tetrahymena pyriformis cells

This study assessed the toxicity of melamine against the unicellular eukaryotic system of Tetrahymena (T.) pyriformis exposed to 0, 0.05, 0.25, 0.5, 2.5, and 5 mg/mL of melamine. Cell growth curves of different cultures, the half maximum inhibition concentration (IC₅₀) value of melamine, and morphological changes in cells were obtained via optical and transmission electron microscopic observation. The effects of eleven melamine concentrations, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 and 5 mg/mL, on protein expression levels of T. pyriformis were examined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The results showed an obvious inhibitory effect of melamine on the growth of eukaryotic cells. Cell growth dynamics indicated that the IC₅₀ value of melamine on T. pyriformis was 0.82 mg/mL. The cellular morphology was also affected in a concentration-dependent manner, with characteristics of atrophy or cell damage developing in the presence of melamine. The relative contents of the top four main proteins corresponding to peak mass-to-charge ratios (m/z) of 4466, m/z 6455, m/z 6514, and m/z 7772 in the MALDI-TOF-MS spectra were all found to be closely correlated with the melamine concentrations. In conclusion, exposure of eukaryotic cells to melamine could inhibit cell growth, cause changes in cytomorphology and even disturb the expression of proteins in a concentration-dependent manner. The described method of examining four sensitive proteins affected by melamine was also proposed to be used in a preliminary study to identify protein biomarkers in T. pyriformis.     

Antimutagenic and anticarcinogenic effect of methanol extracts of Petasites japonicus Maxim leaves

The methanol extract from the leaves of Petasites japonicus Maxim (PJ) was studied for its (anti-)mutagenic effect with the SOS chromotest and reverse mutation assay. The (anti-)carcinogenic effects were evaluated by the cytotoxicity on human cancer line cells and by the function and the expression of gap junctions in rat liver epithelial cell. PJ extracts significantly decreased spontaneous β-galactosidase activity and β-galactosidase activity induced by a mutagen, ICR, in Salmonella (S.) typhimurium TA 1535/pSK 1002. All doses of the extract (0.08-100 mg/plate) decreased the reversion frequency induced by benzo (α)pyrene (BaP) in S. typhimurium TA 98. It decreased not only the spontaneous reversion frequency but also that induced by BaP in S. typhimurium TA 100. PJ extract showed greater cytotoxic effects on human stomach, colon and uterus cancer cells than on other cancer cell types and normal rat liver epithelial cells. Dye transfers though gap junctions were significantly increased by PJ extracts at concentrations greater than 200 µg/mL and the inhibition of dye transfer by 12-O-tetradecanoylphorobol-13-acetate (TPA) was obstructed in all concentrations of PJ. PJ significantly increased the numbers of gap junction protein connexin 43, and increased the protein expression decreased by TPA in a dose-dependent manner. Based on these findings, PJ is suggested to contain antimutagenic and anticarcionogenic compounds.     

Five alternative splicing variants of the TYR gene and their different roles in melanogenesis in the Muchuan black-boned chicken

1. The TYR gene encodes tyrosinase, a multifunctional enzyme that is essential for melanin biosynthesis in melanocytes. This experiment involved the cloning and characterisation of the TYR gene in chicken. Five alternative splice variants were identi?ed in the black feather bulb and designated as TYR-AS1, TYR-AS2, TYR-AS3, TYR-AS4 and TYR-AS5.

2. Among the 11 chicken tissues examined, the feather bulb, comb and skin showed higher levels of all TYR variants. All TYR variants were expressed at significantly different levels in black and white feather bulbs (P < 0.05) and may be involved in melanin formation in plumage. Only TYR-AS1, which plays an important role in muscle melanogenesis, was significantly differentially expressed between black and white muscle (P < 0.01). All TYR variants were expressed at significantly different levels in black and white skin (P < 0.01).

3. The mRNA expression levels of the 5 variants were closely associated with skin melanogenesis in the chicken. These findings provide new clues to the molecular mechanism of melanin formation in the Muchuan black-boned chicken.     

Suppressive effect of culture supernatant of erythrocytes and serum from dogs infected with Babesia gibsoni on the morphological maturation of canine reticulocytes in vitro

The present study evaluated the effects of infected culture supernatant of erythrocytes, fractionation of culture supernatant and serum from dogs infected with Babesia gibsoni (B. gibsoni) on the maturation of canine reticulocytes in vitro. The SDS-PAGE demonstrated that significantly broader bands were generated by both the infected culture supernatant of erythrocytes and the serum from dogs chronically infected with B. gibsoni. The culture supernatant of erythrocytes infected with B. gibsoni strongly suppressed the maturation of reticulocytes. Prior studies showed that chronically infected serum had inhibitory effects on both the maturation of reticulocytes and the canine pyrimidine 5''-nucleotidase subclass I and purine-specific 5''-nucleotidase activity. In addition, serum free infected culture supernatant of erythrocytes had an inhibitory effect on the morphological maturation of reticulocytes. These results suggest that infected serum and culture supernatant of erythrocytes might accumulate excess proteins and/or metabolites as a result of the inhibited maturation of reticulocytes and decreased activity of erythrocyte 5''-nucleotidase. Furthermore, the fractions observed at >150 kDa- and 150-70 kDa- in the infected culture supernatant and serum retarded the maturation of canine reticulocytes in vitro. The results obtained from the in vitro examinations, in the present study, suggested that B. gibsoni itself and/or its metabolites might release certain proteins in the infected culture supernatant and serum from infected dogs and as a result delay morphological maturation of canine reticulocytes.     

Production of monoclonal antibodies against serum immunoglobulins of black rockfish (Sebastes schlegeli Higendorf)

The present study was undertaken to produce monoclonal antibodies (MAbs) against immunoglobulin (Ig) purified from black rockfish (Sebastes schlegeli Higendorf) serum using protein A, mannan binding protein, and goat IgG affinity columns. These three different ligands were found to possess high affinity for black rockfish serum Ig. All of the Igs purified eluted at only 0.46 M NaCl concentration in anion exchange column chromatography and consisted of two bands at 70 kDa and 25 kDa in SDS-PAGE; they also had similar antigenicity for MAbs to Ig heavy chain in immunoblot assays. Therefore, black rockfish Ig is believed to exist as a single isotype within serum. The MAbs produced against Ig heavy chain reacted specifically with spots distributed over the pI range from 4.8 to 5.6 with a molecular weight of 70 kDa on two dimensional gel electrophoresis immunoblot profiles.     

A novel ��-glucan produced by Paenibacillus polymyxa JB115 induces nitric oxide production in RAW264.7 macrophages

The effect of extracellular β-(1→3), (1→6)-glucan, produced by Paenibacillus polymyxa JB115, on nitric oxide (NO) production in RAW264.7 macrophages was investigated. β-glucan induced the production of NO by RAW264.7 macrophages in a concentration- and time-dependent manner. Moreover, β-glucan stimulation increased the mRNA expression of iNOS, COX-2 and IL-6 in RAW264.7 macrophages in a concentration-dependent manner.     

TYRP1基因控制动物色素形成的研究进展

黑色素广泛分布于微生物、植物和动物体内,并具有重要的生理功能。作者综述了黑色素的形成过程并详细分析总结了TYRP1基因特征及其变异对动物肤色的影响,为研究乌骨绵羊和乌骨鸡乌质性状形成的分子基础提供依据。     

Elm tree bark extract inhibits HepG2 hepatic cancer cell growth via pro-apoptotic activity

Control of inflammation is widely accepted as an important strategy for cancer chemoprevention. Anti-inflammatory effects of bark extracts of elm tree (BEE) have been amply reported. Therefore, BEE may be a good candidate cancer chemopreventive agent. Considering the high incidence of hepatic cancer and limited therapeutic approaches for treating this disease, it is important to develop liver cancer-specific chemopreventive agents. To evaluate the chemopreventive potential of BEE, we investigated the growth inhibition effect of BEE on the HepG2 human hepatocellular carcinoma cell line. We performed a cell counting kit-8 assay to determine cell viability, and 4,6-diamino-2-phenylindole staining and flow cytometry to measure apoptotic cell death. Finally, the expression levels of pro- and anti-apoptotic proteins were measured. BEE inhibited the growth of HepG2 cells and induced apoptosis in a dose-dependent manner. Pro-apoptotic activity was promoted via the mitochondrial pathway of apoptosis, as demonstrated by the activation of pro-apoptotic proteins Bax, caspase-9, caspase-3, and poly (ADP-ribose) polymerase as well as the down-regulation of the anti-apoptotic protein Bcl-2. These results suggest that BEE may have potential use in hepatic cancer chemoprevention by suppressing cancer cell growth via pro-apoptotic activity.     

Porphyromonas gulae 41-kDa fimbriae induced osteoclast differentiation and cytokine production

Porphyromonas gulae is considered to be associated with canine periodontitis. We have previously reported that the P. gulae American Type Culture Collection (ATCC) 51700 comprised 41-kDa fimbriae. The purpose of the present study was to demonstrate the roles of 41-kDa fimbrial protein in periodontal disease. In this study, we examined the involvement of the 41-kDa fimbrial protein in osteoclast differentiation and cytokine production in murine macrophages. Furthermore, alveolar bone resorption induced by P. gulae infection in rats was evaluated. To estimate osteoclast differentiation, bone marrow cells and MC3T3-G2/PA6 cells were cultured with or without the 41-kDa fimbrial protein for 7 days. BALB/c mouse peritoneal macrophages were stimulated with the 41-kDa fimbrial protein, and the levels of interleukin (IL)-1β and tumor necrosis factor (TNF)-α production were determined by enzyme-linked immunosorbent assay. Osteoclast differentiation was significantly enhanced by treatment with the 41-kDa fimbrial protein in a dose-dependent manner. The total area of pits formed on the dentine slices with osteoclasts incubated with the 41-kDa fimbrial protein was significantly greater than that of the control. The purified 41-kDa fimbrial protein induced IL-1β and TNF-α production in BALB/c mouse peritoneal macrophages after 6 hr of incubation in a dose-dependent manner. The bone loss level in rats infected with P. gulae was significantly higher than that of the sham-infected rats. These results suggest that P. gulae 41-kDa fimbriae play important roles in the pathogenesis of periodontal disease.     

Equine hyperimmune serum protects mice against Clostridium difficile spore challenge

Clostridium (C.) difficile is a common cause of nosocomial diarrhea in horses. Vancomycin and metronidazole have been used as standard treatments but are only moderately effective, which highlights the need for a novel alternative therapy. In the current study, we prepared antiserum of equine origin against both C. difficile toxins A and B as well as whole-cell bacteria. The toxin-neutralizing activities of the antibodies were evaluated in vitro and the prophylactic effects of in vivo passive immunotherapy were demonstrated using a conventional mouse model. The data demonstrated that immunized horses generated antibodies against both toxins A and B that possessed toxin-neutralizing activity. Additionally, mice treated with the antiserum lost less weight without any sign of illness and regained weight back to a normal range more rapidly compared to the control group when challenged orally with 10⁷ C. difficile spores 1 day after serum injection. These results indicate that intravenous delivery of hyperimmune serum can protect animals from C. difficile challenge in a dose-dependent manner. Hence, immunotherapy may be a promising prophylactic strategy for preventing C. difficile infection in horses.     

Evaluation of anthelmintic antimicrobial and antioxidant activity of <Emphasis Type="Italic">Chenopodium album</Emphasis>

The objective of this study was to evaluate the anthelmintic, antimicrobial and antioxidant activities of Chenopodium album against gastrointestinal nematodes of sheep and some pathogenic microbes. A worm motility inhibition assay was used for in vitro study, and a faecal egg count reduction assay was used for an in vivo study. Various concentrations ranging from 100 to 500 μg/ml of the extract were subjected to antimicrobial screening by disc diffusion method against four selected bacterial (Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas multocida and Escherichia coli) and two fungal (Aspergillus flavus and Candida albicans) strains in order to estimate the medicinal potential of the herb. DPPH (1,1-diphenyl-2-picrylhydrazyl), riboflavin photo-oxidation, deoxyribose, lipid peroxidation assays were used for antioxidant activity. The extracts exhibited dose- and time-dependent anthelmintic effects on the Haemonchus contortus as compared to levamisole. The extract showed maximum inhibitory effect against S. aureus (28 ± 0.14 mm), while as mild inhibitory effect was observed against E. coli among the selected microbial strains. The effect produced by the different extract concentrations was comparable with the standard antibacterial agent streptomycin sulphate and antifungal agent nystatin, which were used as effective positive control in the study. The antioxidant activity showed that the extracts exhibited scavenging effect in concentration-dependent manner on superoxide anion radicals and hydroxyl radicals leading to the conclusion that the plant has broad spectrum anthelmintic, antimicrobial and antioxidant activities and could be a potential alternative for treating various diseases.     

Inhibitory effects of SKF96365 on the activities of K+ channels in mouse small intestinal smooth muscle cells

In order to investigate the effects of {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365 (SKF), which is a non-selective cationic channel blocker, on K⁺ channel currents, we recorded currents through ATP sensitive K⁺ (I_KATP), voltage-gated K⁺ (I_Kv) and Ca²⁺ activated K⁺ channels (I_BK) in the absence and presence of SKF in single small intestinal myocytes of mice with patch-clamp techniques. SKF (10 µM) reversibly abolished I_KATP that was induced by cromakalim (10 µM), which is a selective ATP sensitive K⁺ channel opener. These inhibitory effects were induced in a concentration-dependent and voltage-independent manner. The 50% inhibitory concentration (IC₅₀) was 0.85 µM, which was obviously lower than that reported for the muscarinic cationic current. In addition, SKF (1 µM ≈ the IC₅₀ value in I_KATP suppression) reversibly inhibited the I_Kv that was induced by repetitive depolarizing pulses from −80 to 20 mV. However, the extent of the inhibitory effects was only ~30%. In contrast, SKF (1 µM) had no significant effects on spontaneous transient I_BK and caffeine-induced I_BK. These results indicated that SKF inhibited ATP sensitive K⁺ channels and voltage-gated K⁺ channels, with the ATP sensitive K⁺ channels being more sensitive than the voltage-gated K⁺ channels. These inhibitory effects on K⁺ channels should be considered when SKF is used as a cationic channel blocker.     

The radioprotective effects of the hexane and ethyl acetate extracts of Callophyllis japonica in mice that undergo whole body irradiation

The radioprotective activity of extracts from the red seaweed Callophyllis (C.) japonica was investigated in mice that underwent whole-body exposure to gamma radiation. A methanol extract of C. japonica and its fractions [hexane, ethyl acetate (EtOAc), butanol and the remaining H₂O] were used. Each fraction (100 mg/kg body weight) was administered intraperitoneally (i.p.) 2 times into the BALB/c mice, once at 1 and once at 24 h before exposure to 9 Gray (Gy) of gamma radiation. Pre-irradiation administration of the hexane and EtOAc fractions saved the mice, with their survival rates being greater than 80% at 30 days post-irradiation; the mice that were pretreated with the other fractions showed survival rates lower than 20% over the same time period. To examine the effect of each C. japonica fraction on the survival of intestinal and bone marrow stem cells, the number of intestinal crypts and bone marrow cells in the gamma-irradiated mice were examined. Pre-treatment of mice (i.p., 100 mg/kg body weight at 1 and 24 h before irradiation) with the hexane or EtOAc fraction prior to 6-Gy irradiation significantly protected the number of jejunal crypts and bone marrow cells at 9 days after irradiation. These findings suggest that certain extracts from C. japonica, when they are administered prior to irradiation, play an important role in the survival of irradiated mice, and this is possibly due to the extracts protecting the hematopoietic cells and intestinal stem cells against gamma irradiation.     

GC/MS analysis of high-performance liquid chromatography fractions from Sophora flavescens and Torilis japonica extracts and their in vitro anti-neosporal effects on Neospora caninum

We analyzed alcoholic extracts of herbs possessing anti-neosporal activity against Neospora (N.) caninum. To identify the chemical components of Sophora (S.) flavescens and Torilis (T.) japonica associated with anti-neosporal activity, specific fractions were isolated by high-performance liquid chromatography (HPLC). In vitro activity of the fractions against N. caninum was then assessed. Gas chromatography/mass spectrometry (GC/MS) was used to identify and quantify specific anti-neosporal molecules in the herbal extracts. Almost all HPLC fractions of S. flavescens and T. japonica had higher levels of anti-neosporal activity compared to the not treated control. Active constituents of the extracts were sophoridane, furosardonin A, and tetraisopropylidene-cyclobutane in S. flavescens; 5,17-β-dihydroxy-de-A-estra-5,7,9,14-tetraene, furanodiene, and 9,12-octadecadienoic acid (Z,Z)-(CAS,1) in T. japonica.     

The combined effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and the phytoestrogen genistein on steroid hormone secretion,AhR and ERβ expression and the incidence of apoptosis in granulosa cells of medium porcine follicles

Low doses of endocrine disrupting chemicals (EDCs) used in combination may act in a manner different from that of individual compounds. The objective of the study was to examine in vitro effects of low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 100 pM) and genistein (500 nM) on: 1) progesterone (P₄) and estradiol (E₂) secretion (48 h); 2) dynamic changes in aryl hydrocarbon receptor (AhR) mRNA and protein expression (1, 3, 6, 24 and 48 h); 3) dynamic changes in estrogen receptor β (ERβ) mRNA and protein expression (1, 3, 6, 24 and 48 h); and 4) induction of apoptosis in porcine granulosa cells derived from medium follicles (3, 6 and 24 h). TCDD had no effect on P₄ or E₂ production, but potentiated the inhibitory effect of genistein on P₄ production. In contrast to the individual treatments which did not produce any effects, TCDD and genistein administered together decreased ERβ and AhR protein expression in granulosa cells. Moreover, the inhibitory effect of TCDD on AhR mRNA expression was abolished by genistein. The treatments did not induce apoptosis in the cells. In summary, combined effects of low concentrations of TCDD and genistein on follicular function of pigs differed from that of individual compounds. The results presented in the current paper clearly indicate that effects exerted by low doses of EDCs applied in combination must be taken into consideration when studying potential risk effects of EDCs on biological processes.     

酪氨酸酶转运对黑色素生成影响研究进展

哺乳动物毛色的形成依赖于黑色素细胞中黑色素数量及分布比例,酪氨酸酶类(TYR/TYRP1)的转运及其活性是黑色素合成的关键。衔接蛋白(AP-1/3)和溶酶体相关细胞器生物合成复合物(BLOC-1,2,3)将TYR/TYRP1从早期核内体转运至黑素小体,若TYR/TYRP1无法转运至黑素小体中,黑色素合成则会受到抑制。ATP7A将铜离子转移到TYR/TYRP1,从而激活TYR/TYRP1活性,ATP7A的减少同样会使黑色素细胞中黑色素合成量减少。关注这些复合物在转运TYR/TYRP1的作用及影响其活性的因素,可以从不同的角度解析黑色素合成机理,以此为基础探索哺乳动物毛色的淡化机制,以期为阐明人类黑色素合成障碍的相关疾病提供理论依据。     

Susceptibility to N-methyl-N-nitrosourea-induced retinal degeneration in different rat strains

To evaluate the potential role of genetic background in the susceptibility to retinal degeneration induced by N-methyl-N-nitrosourea (MNU), female rats of the Sprague-Dawley (SD), Long-Evans (LE) and Copenhagen (CH) strains were administered 50 mg/kg MNU or saline at 7 weeks of age. Retina morphology and morphometric analysis of all rats was performed 7 days after MNU administration. Atrophy of both the peripheral and central outer retina occurred in all rat strains exposed to MNU. Decreased photoreceptor cell ratio and increased retinal damage ratio were observed. The severities of the retinal atrophy were similar among all three rat strains. In conclusion, MNU-induced photoreceptor degeneration developed consistently in all three strains regardless of the absence (SD rats) or presence (LE and CH rats) of melanin in the retina, suggesting that genetic and melanin factors did not affect photoreceptor cell death after MNU.