The Lactobacillus gasseri G098 Strain Mitigates Symptoms of DSS-Induced Inflammatory Bowel Disease in Mice.

Inflammatory bowel disease (IBD) is a recurring inflammatory disease of the gastrointestinal tract with unclear etiology, but it is thought to be related to factors like immune abnormalities and gut microbial dysbiosis. Probiotics can regulate host immunity and gut microbiota; thus, we investigated the alleviation effect and mechanism of the strain Lactobacillus gasseri G098 (G098) on dextran sodium sulfate (DSS)-induced colitis in mice. Three groups of mice (n = 8 per group) were included: normal control (NC), DSS-induced colitis mice (DSS), and colitis mice given strain (G098). Our results showed that administering G098 effectively reversed DSS-induced colitis-associated symptoms (mitigating weight loss, reducing disease activity index and pathology scores; p < 0.05 in all cases) and prevented DSS-induced mortality (62.5% in DSS group; 100% in G098 group). The mortality rate and symptom improvement by G098 administration was accompanied by a healthier serum cytokine balance (significant decreases in serum pro-inflammatory factors, interleukin (IL)-6 [p < 0.05], IL-1ß [p < 0.01], and tumor necrosis factor (TNF)-α [p < 0.001], and significant increase in the serum anti-inflammatory factor IL-13 [p < 0.01], compared with DSS group) and gut microbiome modulation (characterized by a higher gut microbiota diversity [p < 0.05], significantly more Firmicutes and Lachnoclostridium [p < 0.05], significantly fewer Bacteroidetes [p < 0.05], and significant higher gene abundances of sugar degradation-related pathways [p < 0.05], compared with DSS-treated group). Taken altogether, our results suggested that G098 intake could mitigate DSS-induced colitis through modulating host immunity and gut microbiome, and strain treatment is a promising strategy for managing IBD.

Analysis of Electrocardiogram Among 108 Patients with Brucella.

OBJECTIVE: To improve our knowledge of cardiac manifestations caused by brucellosis through analyzing abnormal electrocardiograms of patients infected with brucella. METHODS: A total of 108 cases were enrolled, and their electrocardiograms were analyzed and summarized retrospectively. RESULTS: Among 108 cases, 77 (71.3%) had a normal electrocardiogram, and 31 (28.7%) had an abnormal electrocardiogram. There were 13 cases with nodal tachycardia (12%), 9 cases with sinus bradycardia (8%), 7 cases with sinus arrhythmia (6%), 8 cases with left ventricular high voltage (7%), 13 cases with abnormal ST segment and T wave (12%), 2 cases with abnormal Q wave (1.85%), 3 cases with complete right bundle branch block (2.78%), 3 cases with ventricular premature beat (2.78%), 1 case with left anterior fascicular block (0.9%), 1 case with first degree a-v block (0.9%), 1 case with QT internal prolongation (0.9%), 1 case with poor R wave progression (0.9%), and 1 case with short PR interval (0.9%). CONCLUSION: The cardiac manifestations of brucellosis were rare, but the mortality was high. Patients with abnormal electrocardiogram should have improved echocardiography in time. Early detection of the abnormal electrocardiogram could give a hint of cardiac damage to avoid the serious consequences.

Moderate salt treatment alleviates ultraviolet-B radiation caused impairment in poplar plants.

The effects of moderate salinity on the responses of woody plants to UV-B radiation were investigated using two Populus species (Populus alba and Populus russkii). Under UV-B radiation, moderate salinity reduced the oxidation pressure in both species, as indicated by lower levels of cellular H2O2 and membrane peroxidation, and weakened the inhibition of photochemical efficiency expressed by O-J-I-P changes. UV-B-induced DNA lesions in chloroplast and nucleus were alleviated by salinity, which could be explained by the higher expression levels of DNA repair system genes under UV-B&salt condition, such as the PHR, DDB2, and MutSα genes. The salt-induced increase in organic osmolytes proline and glycine betaine, afforded more efficient protection against UV-B radiation. Therefore moderate salinity induced cross-tolerance to UV-B stress in poplar plants. It is thus suggested that woody plants growing in moderate salted condition would be less affected by enhanced UV-B radiation than plants growing in the absence of salt. Our results also showed that UV-B signal genes in poplar plants PaCOP1, PaSTO and PaSTH2 were quickly responding to UV-B radiation, but not to salt. The transcripts of PaHY5 and its downstream pathway genes (PaCHS1, PaCHS4, PaFLS1 and PaFLS2) were differently up-regulated by these treatments, but the flavonoid compounds were not involved in the cross-tolerance since their concentration increased to the same extent in both UV-B and combined stresses.

Mixed-type inhibition of tyrosinase from Agaricus bisporus by terephthalic acid: computational simulations and kinetics.

Tyrosinase inhibition studies are needed due to the agricultural and medicinal applications. For probing effective inhibitors of tyrosinase, a combination of computational prediction and enzymatic assay via kinetics were important. We predicted the 3D structure of tyrosinase from Agaricus bisporus, used a docking algorithm to simulate binding between tyrosinase and terephthalic acid (TPA) and studied the reversible inhibition of tyrosinase by TPA. Simulation was successful (binding energies for Autodock4 = -1.54 and Fred2.0 = -3.19 kcal/mol), suggesting that TPA interacts with histidine residues that are known to bind with copper ions at the active site. TPA inhibited tyrosinase in a mixed-type manner with a K ( i ) = 11.01 ± 2.12 mM. Measurements of intrinsic and ANS-binding fluorescences showed that TPA induced no changes in tertiary structure. The present study suggested that the strategy of predicting tyrosinase inhibition based on hydroxyl groups and orientation may prove useful for screening of potential tyrosinase inhibitors.

[Effect of incubation conditions on the nacre secretion capacity of in vitro epithelium from mantle of Hyriopsis cumingii].

The effects of different calcium ion concentration in culture medium of epithelim, varient pH, and nucleus rotundus present or absent on the calcium metabolism of epithelim from mantle of Hyriopsis cumingii work were investigated in this study. With the variance of calcium ion concentration and pH, the results obtained by using polarizing microscope showed that there was no significant difference on calcium ion containing of epithelim cells between nucleus rotundus present and absent. Meanwhile, the reproduction of epithlim cell in mantle was occurred after incubation and it was further transferred to form pearl sac. The complete pearl sac is formed when connective tissue cells around epithlim cell of pearl sac. An obvious birefringence was observed by using polarizing microscope and its strength was positively affected by the incubation duration and calcium ion concentration of culture medium. The results demonstrated that with the development of cell and tissue of mantle, the absorption of calcium carbonate from culture medium was occurred and there existed the significant correlation between surrounding calcium ion concentration and absorption or secretion of cultures. The largest quantity of calcium sphere was occurred when the calcium ion concentration was 200 ml/L, which was very significantly higher than that of others (p < 0.01). It was also found that the calcium ion absorption was affected by the culture tissue and the status of culture. Higher calcium ion requirement was in primary culture than that in subculture. The calcium ion absorption and secretion was higher in tissue gobbet than that in single cell. A high significant correlation was observed between calcium ion absorption (Y) and pH (X): Y = 52.34 - 5601.23 X (r = 0.9661, p < 0.05). The calcium ion concentration 200mg/L and pH 7.2-7.5 are suggested for incubation conditions of epithelim primary culture in mantle of Hyriopsis cumingii.

Epigenetic modification of histone 3 at lysine 9 in sheep zygotes and its relationship with DNA methylation.

BACKGROUND: Previous studies indicated that, unlike mouse zygotes, sheep zygotes lacked the paternal DNA demethylation event. Another epigenetic mark, histone modification, especially at lysine 9 of histone 3 (H3K9), has been suggested to be mechanically linked to DNA methylation. In mouse zygotes, the absence of methylated H3K9 from the paternal pronucleus has been thought to attribute to the paternal DNA demethylation. RESULTS: By using the immunofluorescence staining approach, we show that, despite the difference in DNA methylation, modification of H3K9 is similar between the sheep and mouse zygotes. In both species, H3K9 is hyperacetylated or hypomethylated in paternal pronucleus relative to maternal pronucleus. In fact, sheep zygotes can also undergo paternal DNA demethylation, although to a less extent than the mouse. Further examinations of individual zygotes by double immunostaining revealed that, the paternal levels of DNA methylation were not closely associated with that of H3K9 acetylation or tri-methylation. Treatment of either 5-azacytidine or Trichostatin A did not induce a significant decrease of paternal DNA methylation levels. CONCLUSION: Our results suggest that in sheep lower DNA demethylation of paternal genomes is not due to the H3K9 modification and the methylated DNA sustaining in paternal pronucleus does not come from DNA de novo methylation.

Some motifs were important for myostatin transcriptional regulation in sheep (Ovis aries).

Many motifs along the 1.2 kb myostatin promoter (MSTNpro) in sheep have been found by the MatInspecter program in our recent study. To further verify the role of the motifs and better understand the transcriptional regulation mechanism of the myostatin gene in sheep, the reporter gene EGFP (enhanced green fluorescent protein) was selected and the wild-type (W) vector MSTNPro(W)-EGFP or motif-mutational (M) vector MSTNPro(M)-EGFP were constructed. The transcriptional regulation activities were analyzed by detecting the fluorescence strength of EGFP in C2C12 myoblasts transfected with the vectors. The results showed that E-box (E) 3, E4, E5 and E7, particularly E3, E5 and E7, had important effects on the activity of the 1.2 kb sheep myostatin promoter. In addition, we also detected several other important motifs such as MTBF (muscle-specific Mt binding factor), MEF2 (myocyte enhancer factor 2), GRE (glucocorticoid response elements) and PRE (progesterone response elements) along the sheep myostatin promoter by the mutational analysis.

Expression of basic fibroblast growth factor results in the decrease of myostatin mRNA in murine C2C12 myoblasts.

During the development and regeneration of skeletal muscle, many growth factors, such as basic fibroblast growth factor (bFGF, FGF-2) and myostatin, have been shown to play regulating roles. bFGF contributes to promote proliferation and to inhibit differentiation of skeletal muscle, whereas myostatin plays a series of contrasting roles. In order to elucidate whether the expression of bFGF has any relationship with the expression of myostatin in skeletal muscle cells, we constructed a eukaryotic expression vector for the expression of exogenous bFGF in murine C2C12 myoblasts. Quantitative RT-PCR assays indicated that with the increase of the expression of exogenous bFGF gene, the expression of endogenous myostatin gene was suppressed at mRNA level and protein level.

[Porcine follistatin cDNA cloning and expression in Escherichia coli].

The total RNA was extracted from porcine ovary. Porcine Follistatin cDNA was cloned by RT-PCR. Complete porcine follistatin cDNA coding sequences are presented including 1038 bp of open reading frame. The purified porcine follistatin cDNA was inserted into pGEX-4T-3 vector to construct the prokaryotic fusion protein expression vector. The recombinant expression plasmid was transformed into BL21 (DE3) and expression was induced by IPTG. Protein products were detected by SDS-PAGE and confirmed by Western blotting analysis, which showed that the yield of the Follistatin cDNA was a 63kD protein expression vector. Follistatin protein was expressed in the form of glutathione-S-transferase (GST) fusion protein in E. coli.

Cloning and sequence analysis of myostatin promoter in sheep.

To better understand the structure and function of the myostatin's gene promoter region in sheep, we cloned and sequenced a 1.517 kb fragment containing the 5'-regulatory region of the sheep myostatin gene (GenBank accession number is AY918121). The promoter sequence consists of three TATA boxes, one CAAT box, and eight putative E-boxes. Some putative muscle growth response elements for Octamer-binding factor 1(Octamer), Activator protein 1(AP1), Growth factor independence 1 zinc finger protein (Gfi-1B), Myocyte enhancer factor 2 (MEF2), Muscle-specific Mt binding site (MTBF), Glucocorticoid response elements (GRE) and Progesterone receptor binding site (PRE) were detected. Some of the motifs are conserved as compared to with that in the goat, bovine and porcine myostatin promoters. However, some differences were also found.

[Secreted expression of porcine interferon-gamma gene in Pichia pastoris].

The porcine interferon-gamma (PoIFN-gamma) gene, in which the sequence encoding signal peptide was replaced by that of the alpha-factor of Saccharomyces cerevisiae, was cloned into Pichia pastoris expression vector pPIC9K. The recombinant plasmid pPIC9K-alpha-PoIFN-gamma was then transformed into Pichia pastoris GS115 cells by electroporation and stable multicopy recombinant Pichia pastoris strains were selected by G418 resistance. Two recombinants of multiple inserts were obtained. SDS-PAGE and Western blot assays of culture broth from a methanol-induced expression strain demonstrated that recombinant PoIFN-gamma, 17kD and 23kD proteins, were secreted into the culture medium. Target proteins, 60% of total proteins, were obtained in the culture medium at the concentration of 108 mg/L. This is the first secreted expression of porcine interferon-gamma gene in Pichia pastoris.

[Normal mice derived from oocytes following intracytoplasmic sperm injection (ICSI)].

This paper describes the use of piezo-driven micropipette for intracytoplasmic sperm injection of mice eggs. The head of fresh spermatozoa from KM (Kunming) fertile mice was individually injected into mature oocytes of hybrid mice B6D2F1. Approximately eighty three percent of sperm-injected oocytes survived, and 84.0% of them fertilized normally (extrusion of the second polar body and formation of male and female pronuclei). The eggs fertilized by sperm injection could develop in vitro to 2-cell (98% vs 94.7%), 4-cell (89.5% vs 92.1%) stages, no significantly (P > 0.05) different from embryos fertilized in vivo but there were significantly (P < 0.01) few morulae (63.8% vs 84.2%) and blastocysts (25.7% vs 68.4%) developed in vitro after further culture in vitro in the group of ICSI. When 120 embryos at the pronuclear stage were transferred to seven pseudopregnant KM female, 23.3% of the embryos (0 - 50%, depending on the host) reached the full term. Except for three that were cannibalized soon after birth, all of the young (25 pups) developed into normal and fertile adult. Here we report the first birth of mouse offspring following ICSI in China. These studies may increase understanding of the fertilization process and of how ICSI works.

[Study on effect of Lactobacillus acidophilus MG2-1 on serum lipid metabolism in rats].

Wistar rats were fed with a high lipid diet supplemented with living or thermal death bacteria of Lactobacillus acidophilus MG2-1 which was isolated from koumiss in Mongolia and was of good ability of acid tolerance and decreasing the level of cholesterol in vitro. The effect of Lb. acidophilus MG2-1 on the metabolism of serum cholesterol was discussed. It was showed that it was on the 14th day of experiment that the inhibiting effects of the increase of serum cholesterol level of rat groups fed with living bacteria and heat-killed bacteria was significantly (p > 0.05) and very significantly (p < 0.01) higher than that of the high lipid diet group respectively; at the same time, the level of serum HDL-C of the thermal death bacteria group was significantly higher than that of the high lipid diet group (p < 0.05), also arteriosclerosis index of wistar rats in experimental group is significantly lower than that of the high lipid diet group (p < 0.01). The total bile acid level of the thermal death bacteria group in fecal is significantly higher than that of the high lipid diet group (p < 0.05). It is suggested that the increase of serum cholesterol level in rats can be inhibited and arteriosclerosis can also be prevented by this strain. During the period of tests, the effect of the strain on serum lipid in rats weaken with the time going, while the dose of bacteria fed was not changed.

[Molecular cloning, expression mutation of myostatin and study on biochemical activity of its C-terminal peptide].

Myostatin, a member of the TGF-beta family, negatively regulates skeletal muscle development. Mutation of myostatin activity leads to increases muscle growth and carcass lean yield. The bovine myostatin mutation cDNA was amplified by polymerase chain reaction, and then sub-cloned into the expression vector pET-30a( + ) to form the expression plasmid pET30a (+)-action/ Myostatin. The recombinant plasmid was transformed into E. coli BL21. The overexpression product of pET30a (+)-action/ Myostatin was been showed in vitro. Sheep skeletal muscle cell were cultured with the purified myostatin mutation C-terminal peptide. The results of this study suggest that had a powerful activity to stimulate the hyperplasia and proliferation of sheep muscle cells and shows high biochemical activity.

[Transgenic animals bioreactors].

The production of human recombinant proteins in milk of transgenic farm animals offers a safe, very cost-effective source of commercially important proteins that cannot be produced as efficiently in adequate quantities by other methods. This review has summarized the current status of gene selection, vector construct, transgenic methods, economics, and obvious potential in transgenic animals bioreactors. Recently, a more powerful approach was adopted in the transgenic animals founded on the application of nuclear transfer. As we will illustrate, this strategy presents a breakthrough in the overall efficiency of generating transgenic farm animals, product consistency, and time of product development. The successful adaptation of Cre-/lox P-mediated site-specific DNA recombination systems in farm animals will offer unprecedented possibilities for generating transgenic animals.

[Sheep transgenic embryos produced by intracytoplasmic sperm injection].

In this study, the possibility of sheep transgenesis by intracytoplasmic sperm injection (ICSI) was assessed. In experiment 1, activation of ovine oocytes matured in vitro in preparation for ICSI has been investigated with 3.42 mmol/L Ca2+ treatment, ionomycin alone and ionomycin followed by 6-dimethylaminopurine (DMAP) after 3-h delay (group 1, 2 and 3, respectively). After activation, the oocytes were then cultured in SOFaaBSA medium. Cleavage rates were significantly (P<0.05) different among three groups (18.4%, 91.8% and 71.7%, respectively). In additional culture, no parthenotes in group 1, whereas 11% and 17.4% in group 2 and 3 developed to the blastocyst stage. Therefore we used the third activation method in the following ICSI tests. In experiment 2, development of ovine oocytes after ICSI was investigated. Thawed semen from two rams was separated by Percoll centrifugation and was used for ICSI or in vitro fertilization (IVF) trails. A total of 71.8% of oocytes reached the 2-cell stage following living sperm injection, which was significantly (p>0.05) different from those following IVF (41.4%) and sham-ICSI (30.2%). After seven days' culture, no sham-injected oocytes developed into the blastocyst stage, although 7% in ICSI and 16.1% in IVF-oocytes developed into the blastocyst stage, but there was no significant difference in ICSI and IVF groups (p>0.05). In the further study, the possibility of sheep transgenesis by ICSI was assessed. After coinjection of ovine oocytes matured in vitro with dead sperm cold to -20 degrees C and exogenous DNA encoding green fluorescent protein (GFP), seventy-three percent of coinjected oocytes developed to 2-cell stage (33/45) and two of them were transgene-expressing embryos. Among ten embryos at the 16-cell stage, all embryonic cells in one transgenic embryo still expressed GFP. Four coinjected blastocysts were thawed and transferred to the uterine of the two progesterone-synchronized recipient ewe. No pregnancies were detected on the 60th day. These results suggested sheep transgenic embryos could be produced by ICSI and further studies should be performed.