[Molecular structure and alternative splicing analysis of heat shock factors of Schistosoma japonicum].

OBJECTIVE: To clone and identify the heat shock factors (HSFs) of Schistosoma japonicum and analyze its molecular structure and alternative splicing pattern. METHODS: The New Zealand rabbits were infected with the cercariae of Schistosoma japonicum and were killed and dissected 42 days post-infection, and the adult worms of S. japonicum and the livers of the rabbits were harvested. Then, the total RNA was extracted by using Trizol reagent. The Sj-hsf open reading frame (ORF) and the alternative splicing fragments were amplified by RT-PCR from the female, male and egg samples, then cloned and verified by enzyme digestion and sequencing. DNAMAN 8.0, InterPro, Mega 6 combined with the Internet databases were utilized to clarify the gene structure, functional domains, alternative splicing pattern, and the homology and phylogenetic tree of HSFs. RESULTS: Sj-hsf ORF and the alternative splicing fragments were amplified from the female, male and egg samples of S. japonicum by RT-PCR. After cloning, the positive recombinant plasmids pBSjHSFf-F, pBSjHSFf-M, pBSjHSFf-E containing Sj-hsf ORF, pBSjHSFs-F, pBSjHSFs-M, pBSjHSFs-E with Sj-hsf alternative splicing fragments were identified by enzyme digestion and sequencing. Three alternative splicing Sj-hsf isoforms were observed through sequence analysis: Sj-hsf-isoform1 (2 050 bp), Sj-hsf -isoform2 (2 086 bp) and Sj - hsf -isoform3 (2 111 bp); the GenBank accession numbers were KU954546, KX119143 and KX119144, respectively. All the three isoforms located in the same Contig SJC_S000780 of S. japonicum genome and all expressed at female, male and egg stages, but Sj-hsf-isoform1 with a high-level expression. Sj-HSF-isoform1 (671 aa) and Sj-HSF-isoform2 (683 aa) had DBD (DNA binding domain), HR-A/B and HR-C domains, while Sj-HSF-isoform3 (282 aa) stopped in advance without HR-C domain. Phylogenetic tree analysis of HSFs illustrated that Sj - HSFs belonged to HSF1 family, with a close phylogenetic relationship to Sm-HSFs. CONCLUSIONS: There are three alternative splicing isoforms of Sj-HSF existing in the female, male and egg stages of S. japonicum, but Sj-HSF-isoform1 expresses in a high-level. This study lays the foundation for further study on molecular mechanisms of Sj-HSFs in regulating the heat shock response system.

DNA methylation of ZIC1 and KLOTHO gene promoters in colorectal carcinomas and its clinicopathological significance / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To determine DNA methylation status of ZIC1 and KLOTHO gene in colorectal carcinomas and its relationship with clinicopathological features of patients.</p><p><b>METHODS</b>The mRNA expression of ZIC1 and KLOTHO genes in colorectal carcinomas was detected by real-time quantitative RT-PCR, and the promoter methylation status was detected by methylation specific PCR (MSP). The relationship of ZIC1 and KLOTHO methylation status with clinicopathological features of colorectal carcinoma was analyzed.</p><p><b>RESULT</b>The mRNA expression levels of ZIC1 and KLOTHO genes were significantly down-regulated in tumor tissues when compared to adjacent nontumor tissues (P<0.001). ZIC1 and KLOTHO methylation was detected in 80.0%(20/25) and 76.0%(19/25) of colorectal tumor tissues, respectively, and the both positive rate was 64.0%(16/25).</p><p><b>CONCLUSION</b>The down-regulated expression of ZIC1 and KLOTHO in colorectal carcinoma may relate to promoter methylation. The detection of methylation of ZIC1 and KLOTHO gene potentially provides biomarkers for diagnosis of colorectal carcinoma.</p>

Detection of HER-2 mRNA and protein expression in breast cancer tissues by using chromogenic in situ hybridization and immunohistochemistry methods / 肿瘤

Objective: To detect human epidermal growth factor receptor-2 (HER-2) gene amplification and protein expression by using chromosome in situ hybridization (CISH) method and compare the results with immunohistochemical assays (IHC). Methods: We assessed the amplification status of HER-2 gene with CISH method in 145 cases of breast cancer tissues. Fourteen out of 145 specimens have been tested by fluorescence in situ hybridization (FISH). HER-2 gene amplification and protein expression in the 145 cases of breast cancer tissues were tested by IHC methods. We retrospectively compared the detection results among the three methods and analyzed the correlation of HER-2 expression with various high-risk factors of breast cancer such as pathological grade, lymph node metastasis, menopausal status, and the expression of estrogen receptor (ER)/progesterone receptor (PR). Results: Based on CISH assay, non-amplification of HER-2 gene was observed in 71 cases (50.0%), low-amplification was detected in 11 cases (7.6%), and high-amplification was assessed in 63 cases (43.4%). The concordance rate between FISH (n = 14) and CISH was 100.0% (14/14). The concordance rate between IHC and CISH was 84.8% (n = 145, P 90%) was in agreement with its protein expression. However, the amplification rate of HER-2 gene was only 61.1% IHC + + cases. Both CISH and IHC detection demonstrated that expression of ER/PR was negatively related with the expression of HER-2. The amplification rate of HER-2 gene was significantly higher in ER/PR-negative patients that that in ER/ PR-positive patients (CISH: 68.3% vs 38.8%, P < 0.01; IHC: 71.7% vs 48.2%, P < 0.01). The amplification status of HER-2 gene had no correlation with tumor pathological grade, auxiliary lymph node metastasis, and menopausal status. Conclusions: CISH for HER-2 detection is easy to handle and has high accuracy. It could replace FISH to further confirm the status of HER-2 gene in IHC + + cases. HER-2 is negatively related with ER/PR expression but has no relationship with other risk factors such as pathological grade, auxiliary lymph node metastasis, and menopausal status. HER-2 should be tested independently.

Effect of mucosal protective on the quality of gastric ulcer healing / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To explore the mucosal protective effect on the quality of gastric ulcer healing.</p><p><b>METHODS</b>Gastric ulcers were induced in male rats by serosal application of acetic acid. Rats were gavaged for 14 days with saline, omeprazole (OME), teprenone (TEP) and TEP plus OME starting 3 days after ulcer induction. Then the tissues and blood samples were obtained and measured.</p><p><b>RESULT</b>The lower ulcer index (UI) and increased ulcer inhibition rate were observed in OME and OME+TEP groups. In TEP and OME+TEP groups, restored mucosa thickness increased, cystically dilated glands decreased, microvessels in connective tissue increased, the secretion of mucus, hexosamine, PGE(2), bFGF were enhanced, the expression of EGFR was increased.</p><p><b>CONCLUSION</b>TEP can improve the quality of gastric ulcer healing, when combined with OME,the effect is more marked.</p>

A probiotic treatment containing Lactobacillus, Bifidobacterium and Enterococcus improves IBS symptoms in an open label trial / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>To evaluate the efficacy and safety of live combined Bifidobacterium, Lactobacillus and Enterococcus capsules in treatment of irritable bowel syndrome.</p><p><b>METHODS</b>Eighty-five patients [male 32, female 53; age (45.31+/-11.72) years] were given live combined Bifidobacterium, Lactobacillus and Enterococcus capsules 1260 mg/d t.i.d.x4 weeks. Syndrome scales were used to evaluate the efficacy in gastrointestinal syndrome. Fecal flora was also measured before and after the treatment. Six bacteria were cultured and the colony forming units were counted in stool. SPSS was used for data analysis.</p><p><b>RESULTS</b>Seventy-four patients finished the follow-up. No side-effect was found. For treatment of irritable bowel syndrome, the effective rate of live combined Bifidobacterium, Lactobacillus and Enterococcus capsules was 56.8% in the second week, 74.3% in the fourth week and 73.0% in the sixth week. Single symptom was improved, especially in abdominal pain and stool character. The probiotica containing live combined Bifidobacterium, Lactobacillus and Enterococcus could increase bifidobacterium count (P<0.01) and lactobacillus count (P<0.05); decrease bacteroides count (P<0.05) and enterococci count (P<0.01); No obvious changes were observed in clostridium difficile colonitis and enterobacteriaceae (P>0.05).</p><p><b>CONCLUSION</b>The result of the study indicated that the administration of live combined Bifidobacterium, Lactobacillus and Enterococcus improved the symptom of irritable bowel syndrome and that there was a gradual increase of this effect. Thereafter conditions remained stable for 2 weeks. That improvement may be associated with alterations in gastrointestinal flora.</p>

Morphological and pathologic changes of experimental chronic atrophic gastritis (CAG) and the regulating mechanism of protein expression in rats / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>To study the pathologic change and molecular regulation in cell proliferation and apoptosis of gastric mucosa in rats with chronic atrophic gastritis (CAG), and evaluate the possible mechanisms.</p><p><b>METHODS</b>Rats were administered with 60% alcohol or 2% salicylate sodium, 20 mmol/L deoxycholate sodium and 0.1% ammonia water to establish chronic atrophic gastritis (CAG) models. The gastric specimens were prepared for microscopic view with hematoxylin and eosin (H-E) and alcian blue (A-B) stain. The number of infiltrated inflammatory cells, the thickness of the mucosa gland layer (microm) and the number of gastric glands were calculated. The damage of barrier in mucosa with erosion or ulceration, and the thickness of mucin were examined by scanned electron microscope (SEM). The levels of PGE(2), EGF (epiderminal growth factor) and gastrin in the serum were measured with radioimmunoassay or ELISA method. The immunohistochemistry method was used to observe the number of G cells, the expression of protein of EGFR (EGF receptor), C-erbB-2, p53, p16 and bcl-2 in gastric tissue.</p><p><b>RESULTS</b>Under SEM observation, the gastric mucosa was diffused erosion or ulceration and the thickness of mucin was decreased. Compared with normal rats, the grade of inflammatory cell infiltration in CAG rats was elevated, whereas the thickness and number of gastric gland were significantly lower (P<0.05). Compared with normal level of (0.61+/-0.28) microg/L, EGF in CAG (2.24+/-0.83) microg/L was significantly higher (P<0.05). The levels of PGE(2) and gastrin in serum were significantly lower in CAG rats than that in normal rats (P<0.05). Immunohistochemistry detection showed that the number of G cell in antrum was lower in CAG group (P<0.05). Immuno-stain showed EGFR protein expression in the basal and bilateral membrane, and the cytoplasma in atrophic gastric gland, while negative expression was observed in normal gastric epithelial cells. Positive staining of p53 and p16 protein was localized in the nucleus of epithelial cells. The former was higher positively expressed in atrophic gland, while the later was higher positively stained in normal gastric tissue. bcl-2 protein was positively stained in the cytoplasma in atrophic gastric gland, while very weakly stained in normal gastric tissue.</p><p><b>CONCLUSION</b>The pathological findings in gastric gland accorded with the Houston diagnostic criteria of antrum-predominant CAG. CAG in rats was related with the damage of barrier in gastric mucosa and the misbalance of cell proliferation and apoptosis. There was high protein expression of oncogene, while inhibitor of suppressor gene in CAG rats indicated high trend of carcinogenesis.</p>

Intestinal mucosa protection of muscovite n ulcerative colitis in rats / 中国中药杂志

<p><b>OBJECTIVE</b>To examine the efficacy of Muscovite on acetic acid-induced ulcerative colitis in rats, and to research the mechanisms of intestinal mucosal protection.</p><p><b>METHOD</b>Ulcerative colitis was induced in rats by intracolonic injection of 2 mL of 7% acetic acid. Rats were treated with three different doses of the Muscovite and SASP at random by intracolonic injecion, the normal saline was considered as control group. The rats were sacrificed and the colons were excised and opened longitudinally. Under a dissecting microscope, gross findings were observed and scored. MPO activity was assayed by spectrophotometry in colonic mucosa.</p><p><b>RESULT</b>Gross finding showed that multiple ulcer with diameter more than 1 cm, surrounded with erosion, erythematous and edema in the proximal colon in ulcerative coltis. The colon from Muscovite treatment group were histopatholgically normal, with slight erosion, erythematous and edema. The colon in SASP group had small ulceration and severe erosion and edema. The score of gloss change were significant lower in Muscovite groups than that in normal saline group (P < 0.01). There were necrosis and exfoliation of mucosa, multiple cystic dilation of mucosa gland, and large number of and inflammation attenuated in Muscovite groups. There nerutrophils and vessel infiltration in ulcerative colitis. The ulceration disappeared were erosion in mucosa and inflammatory cell infiltrating into submucosa in SASP group. Compared with normal saline group, the pathological scale were significant decreased in Muscovite and SASP groups (P < 0.05). The MPO activity was significant increased in colitis tissue compared with normal group (P < 0.001). After administrating with Muscovite or SASP, the level of MPO were significant decreased (P < 0.01).</p><p><b>CONCLUSION</b>Muscovite has the effect of mucosal protection by attenuating the inflammation of colonic mucosa and decreasing the activity of MPO.</p>

Mechanisms of muscovite on gastric mucosal protective effect / 中国中药杂志

<p><b>OBJECTIVE</b>To explore the mechanisms of muscovite gastric mucosal protective effect.</p><p><b>METHOD</b>Rat model of chronic gastritis were used. After gastric mucosal injury was induced, the rats were divided into 6 groups and were treated with different drugs. 2 weeks later, the tissue and blood samples were obtained and measured.</p><p><b>RESULT</b>The general conditions, the observations under macroscopy, microscope and electron microscope of the middle and high dose of muscovite groups resembled those of the normal group. Their PH levels were higher than those of the model group, and the rates of intestinal metaplasia were lower, but the PGE2 level of the middle dose of muscovite group was the highest.</p><p><b>CONCLUSION</b>Muscovite can be adsorbed on the surface of the gastric mucosa. It has gastric mucosal protective effect by improving excretion of mucus and synthesis of PGE2 in gastric mucosa, restraining gastric acid, reversing of intestinal metaplasia and decreasing inflammation cells.</p>