Objective: To facilitate the functional analysis of chromosomal genes and their products, the recombineering technique to epitope tagging of chromosomal genes of Y. pestis was adapted. Methods: The epitope tag was generated by primer annealing and then fused with resistance gene by fusion PCR. The epitope-resistance cassette was inserted into pBluecript, resulted in the template plasmid, pBS-MH. The tagging cassette for rpoS was obtained by PCR amplification from pBS-MH with primers containing homology specific to the target gene. PCR products were transformed into recombination competent cells and recombinants were selected. PCR and DNA sequencing were used to confirm the correct tagging event. The expression of the tagged protein was detected with Western blot by using monoclonal antibody to the epitope. Results: The template plasmid containing fusion of epitope and resistance gene was successfully constructed. The sigma factor gene, rpoS, was tagged with a myc-his tag at the COOH terminus. Expression of the tagged rpoS was successfully detected indirectly by the antibody against His tag. Conclusion: The chromosomal gene tagging by recombineering technique represents a powerful tool in the functional study of bacterial genes and their products.

OBJECTIVETo evaluate the clinical prosthetic effect of IPS-Empress 2 pressahie ceramic crowns.

METHODS198 teeth of 70 patients were restored with IPS-Empress 2 pressahie ceramic crowns. The patients were asked to return in one week and every half year. The clinical prosthetic effect was evaluated.

RESULTSThrough follow-up of 3-38 months, the veneer porcelain crowns of 3 teeth were broken. 2 crowns fall off due to teeth fracture, gingivitis occurred in 2 teeth, pulpitis or periapical periodontitis occurred in 3 teeth. The shades of 3 crowns were darkening. The prosthetic effect of 185 teeth was satisfied. The rate of satisfaction was 93.4%.

CONCLUSIONIPS-Empress 2 pressable all-ceramic crown has the advantages of aesthetic effect, good hiocompatihility and simple fabrication. But its strength is not enough for posterior teeth and it can not cover the deep color of non-vital teeth and metal materials.

Aluminum Silicates ; Ceramics ; Crowns ; Dental Porcelain ; Humans ; Lithium Compounds

Objective To study the clinical characteristics and digital subtract angiography (DSA) manifestations of the hemorrhagic moyamoya disease in adults. Methods A retrospective study was conducted to analyze the clinical data and DSA imaging of 68 adult patients who had been diagnosed with intracerebral hemorrhage on CT and with moyamoya on DSA. Results They were 23 males and 45 females with an average of 37.8 years. Their DSA manifestations included bilateral lesions on the anterior cerebral circulation in 57 cases, 19 of whom had combined lesions on the posterior cerebral circulation; unilateral lesion on the anterior cerebral circulation in 9 cases, 6 of whom had combined lesions on the posterior cerebral circulation; simple lesions on the posterior cerebral circulation in 2 cases;simple lesions on the anterior cerebral circulation in 41 cases.Intracranial aneurysm was complicated in 11 cases, 7 of whom underwent aneurysm embolization or clipping of the aneurysm neck. Cerebral infarction was complicated in 13 cases. Repeated hemorrhage for twice or more occurred in 17 cases.Ventricle hemorrhage occurred in 59 cases, parenchymal hemorrhage in 7 cases, and subarachnoid hemorrhage in 2 cases. Craniotomy was performed in 6 cases of massive intracranial bleeding.Conclusions Adult patients with hemorrhagic moyamoya disease, particular middle-aged women,usually have a major manifestation of intraventricular hemorrhage. Surgical interventions for combined aneurysms and symptoms can achieve a good therapeutic effect, but it is difficult to prevent re-hemorrhage.DSA is the primary method to determine specific characteristics of moyamoya lesions and consequently to choose a proper treatment.

OBJECTIVETo evaluate the fast serum pepsinogen level of the healthy adults among local population in areas with high incidence of gastric cancer and to study the suitable cut-off values of serum pepsinogen abnormality for the screen of chronic atrophic gastritis (CAG) and gastric carcinoma (GC) in China.

METHODSSerum PG I and PG II levels were detected with time resolved fluorescence immunoassay (TRFIA). The fast serum PG I and PG I level as well as PG I/PG II ratio of 606 healthy adult residents among local population in Zanhuang county, Hebei province were detected and the normal distribution ranges determined. The relationship between different cut-off values of serum PG I level, PG I/PG II ratio and corresponding pathological changes in gastric mucosae were comparatively analyzed with serum PG detection, endoscopic biopsy and pathological observation in 720 cases of local residents receiving endoscopic examination in the high incidence area of gastric cancer. The efficacy, sensitivity and specificity of different PG I, PG II abnormality cut-off values in the screen p rogram of CAG and GC were statistically analyzed.

RESULTSThe serum PG I, PG II and PG I/PG II ratio levels of healthy adults from a local natural population in the high incidence area of gastric cancer were all skewed from normal distribution. The median level of PG I, PG II and PG I/PG II were 161 microg/L, 14.8 microg/L and 10.5 respectively. Data from comparative studies on serum PG level and pathological changes of gastric mucosae showed that within the serum PG I range from 40 microg/L to 80 microg/L and PG I/PG II ratio range from 3 to 8, sensitivity of the screening program for CAG and GC increased while the specificity decreased along with the increase of cutoff values of serum PG I and PG I/PG II ratio. Results from statistical receiver operator characteristic curve (ROC) analysis suggested that the best cut-off value of PG I and PG I/PG II abnormality for the screening of CAG and GC being PG I < or =60 microg/L,PG I/PG II < or =6 respectively.

CONCLUSIONThe serum PC I, PG II and PG I/PG II ratio levels of healthy adults from a local natural population in the high incidence area of gastric cancer were all skewed from normal distribution. Serum PG I < or =60 microg/L and PG I/PG II ratio < or =6 as abnormal cut-off value for the screen of CAG and GC could result relatively good sensitivity and specificity.

China ; Chronic Disease ; Gastritis, Atrophic ; blood ; diagnosis ; Humans ; Mass Screening ; Pepsinogen A ; blood ; Reference Values ; Sensitivity and Specificity ; Stomach Neoplasms ; blood ; diagnosis

OBJECTIVETo study the correlation between serum pepsinogen (PG) level and gastric mucosal changes of the residents who live in the high incidence area of gastric cancer, and investigate the value of serum PG level in screening for chronic atrophic gastritis (CAG) and gastric cancer (GC).

METHODSSerum PG level was detected with time resolved fluorescence immunoassay (TRFIA). The correlation between serum PG level and gastric mucosal changes was analyzed through endoscopic biopsy and pathological examination in 720 adult residents.

RESULTSThe median serum PG I, PG II level and PG I / PG II ratio in 30 healthy residents with normal gastric mucosa was 172.0 microg/L, 9.6 microg/L and 17.5, respectively. The median serum PG I level of GC patients was significantly lower than that of chronic gastritis patients, gastric ulcer (GU) patients and local healthy residents (P < 0.05). The median PG I level of GU patients was significantly higher than that of the healthy resident group and the other groups (P <0.05). Serum PG II level in CAG, GC and GU groups were all significantly higher than that in CSG and healthy resident group (P <0.05). The PG I/PG II ratio in CAG or GC patients was significantly lower than that in the other groups (P < 0.05). The sensitivity and specificity of serum PG I < or = 60 microg/L for screening CAG or GC was 19.7% and 95.5% respectively, which were 34.7%, 89.3% for PG I/PG II < or =6, and 14.1%, 97.3% for PG I < or =60 microg/L + PG I /PG II < or =6. None in GU group was found to have serum PG I < or =60 microg/L. The median serum PG I level and PG I /PG II ratio in chronic gastritis (including CSG and CAG) with intestinal metaplasia were significantly lower than that of healthy resident group (P < 0.05). The sensitivity and specificity for screening of intestinal metaplasia were 16.6% and 92.9% by PG I < or =60 microg/L; 25.6% and 80.4% by PG I/PG II < or =6; 11.9% and 93.9% by PG I < or =60 microg/L + PG I/ PG II < or = 6.

CONCLUSIONSerum pepsinogen level of the residents in the high incidence area of gastric cancer is closely correlated with the pathological changes of gastric mucosa. Though the sensitivity of serum pepsinogen level is relatively lower in the screening for chronic gastritis, gastric cancer and intestinal metaplasia, the specificity was quite high. PG I < or = 60 microg/L may be usful in differential diagnosis of gastric cancer from gastric ulcer.

Diagnosis, Differential ; Gastric Mucosa ; pathology ; Gastritis, Atrophic ; blood ; diagnosis ; pathology ; Humans ; Metaplasia ; Pepsinogen A ; blood ; Pepsinogen C ; blood ; Sensitivity and Specificity ; Stomach Neoplasms ; blood ; diagnosis ; pathology ; Stomach Ulcer ; blood ; diagnosis ; pathology

OBJECTIVETo investigate the clinicopathological characteristics of colorectal neuroendocrine neoplasms (NENs) and the prognostic significance of the new WHO classification and staging system about gastroenteropancreatic NENs.

METHODSThe clinical and pathological records were reviewed in 73 patients with colorectal NENs (carcinoids). All slides were retrieved and reviewed, immunohistochemical staining (EnVision method) was performed and follow-up information retrieved.

RESULTSForty-one men and thirty-two women were included with a median age of 53 years (19 - 79 years). The location of the primary tumors in 65 patients was within 10 cm from the anorectal line. In 45 cases, the tumor diameter was ≤ 1 cm (no metastasis occurred); in 11 cases, the tumor diameter was > 1 cm but ≤ 2 cm (two patients had metastatic tumors); in 17 cases, the tumor diameter was > 2 cm (12 patients had metastatic tumors). The metastatic rate was significantly correlated with tumor size (P = 0.000). All tumors were immunoreactivity for synaptophysin and/or chromogranin A. According to the criteria of WHO classification and staging system about gastroenteropancreatic NENs, there were 65 cases of neuroendocrine tumors, including 51 cases of grade 1 (G1), 14 cases of grade 2 (G2), 4 cases of neuroendocrine carcinoma (G3) and 4 cases of mixed adenoneuroendocrine carcinoma. Following-up data showed that of the 34 patients with G1 tumor, there were no tumor-related death, but two patients showed metastases, and the remaining patients were disease free for 6 to 179 months. Of the 12 patients with G2 tumors, five developed metastasis, there were two tumor-related deaths, and the nine surviving patients were alive for 17 to 118 months. Of the four G3 patients, all developed metastasis and there were three tumor-related deaths. Of the four mixed adenoneuroendocrine carcinoma there were two tumor-related deaths. The difference of metastatic rate, tumor-related mortality, and overall survival among different grading groups in this series was statistically significant (P = 0.000).

CONCLUSIONSColorectal neuroendocrine neoplasm is a group of tumors with distinct prognostic difference, and most of these tumors show an indolent clinical behavior. There is a good correlation between the new WHO classification and staging system of gastroenteropancreatic NENs and their clinical behaviors.

Adult ; Aged ; Carcinoma, Neuroendocrine ; metabolism ; pathology ; radiotherapy ; secondary ; surgery ; Chromogranin A ; metabolism ; Colorectal Neoplasms ; metabolism ; pathology ; radiotherapy ; surgery ; Female ; Follow-Up Studies ; Humans ; Liver Neoplasms ; secondary ; Male ; Middle Aged ; Neoplasm Grading ; Neoplasm Invasiveness ; Neuroendocrine Tumors ; metabolism ; pathology ; radiotherapy ; secondary ; surgery ; Survival Rate ; Synaptophysin ; metabolism ; Tumor Burden ; Young Adult

In order to study the important factors involved in cationic liposome-mediated gene transfer, Lipofectamine 2000 or DOTAP was evaluated using three types of cells (Hep-2, MCF-7 and SW-480) in vitro transfection efficiencies. Different properties of the two reagents were analyzed and compared by DNA arrearage assay and MTT assay. Both Lipofectamine 2000 and DOTAP had strong capability to combine with DNA; Lipofectamine 2000 can get higher transfection efficiency of the three cells by using GFP as report gene, meanwhile, DOTAP can also get higher transfection efficiency against Hep-2 cell. However, DOTAP showed lower transfection efficiency against MCF-7 and SW-480 cell. On the other hand, the cytotoxicity assay showed that over 85% cell viability of MCF-7 cell could be achieved both by Lipofectamine 2000 and DOTAP under the optimal transfection condition. Relatively speaking, Lipofectamine 2000 has very high transfection efficiency in a broad range of cell lines, but because of the special selectivity of cell type on liposome, DOTAP also has a broad application prospect.
Cell Line, Tumor ; Cell Survival ; drug effects ; DNA ; genetics ; Fatty Acids, Monounsaturated ; chemistry ; toxicity ; Gene Transfer Techniques ; Genes, Reporter ; Genetic Vectors ; Green Fluorescent Proteins ; metabolism ; Humans ; Lipids ; chemistry ; toxicity ; Quaternary Ammonium Compounds ; chemistry ; toxicity ; Transfection

OBJECTIVEThis study examined the effects of maternal deficiency of folic acid during pregnancy on pulmonary development and protein A (SP-A) expression in newborn rats in order to explore the possible mechanism of lung developmental disorders.

METHODSThirty-six adult Sprague-Dawley female rats were randomly assigned into two groups: control and study (n=18). The study and the control groups were fed with fodder containing folic acid or not respectively. Two weeks later, the female rats in the two groups copulated with normal male rats. Newborn rats were sacrificed at 1, 7 and 14 days after birth (8 pups at each time point). Lung sections were stained with hematoxylin and eosin for histological examination. SP-A expression of protein and mRNA were determined by immunohistochemistry and real-time quantitative RT-PCR, respectively.

RESULTSThe newborn rats from the study group showed damaged lung tissue structures. The mean optical density of type II cells with positive expression of SP-A decreased significantly from 1 to 14 days in newborn rats of the study group compared with the control newborn rats (P<0.05). The real-time quantitative RT-PCR showed that the expression of lung SP-A mRNA also decreased significantly from 1 to 14 days in newborn rats of the study group compared with control newborn rats (P<0.05).

CONCLUSIONSMaternal deficiency of folic acid during pregnancy can decrease the expression of SP-A in lung tissues of newborn rats, which might lead to the disorder of lung development maturation.

Animals ; Animals, Newborn ; Female ; Folic Acid Deficiency ; metabolism ; Immunohistochemistry ; Lung ; embryology ; Male ; Pregnancy ; Pregnancy Complications ; metabolism ; Pulmonary Surfactant-Associated Protein A ; analysis ; genetics ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction

Acquired Immunodeficiency Syndrome ; epidemiology ; Adult ; China ; epidemiology ; Cross-Sectional Studies ; Female ; HIV Infections ; epidemiology ; Humans ; Middle Aged ; Poverty Areas ; Young Adult

Stolon is an important organ for reproduction and regeneration of Amana edulis. Previous analysis of transcriptome showed that MYB was one of the most active transcription factor families during the development of A. edulis stolon. In order to study the possible role of MYB transcription factors in stolon development, the authors screened out an up-regulated MYB gene named AeMYB4 was by analyzing the expression profile of MYB transcription factors. In the present study, sequence analysis demonstrated that AeMYB4 contained an open reading frame of 756 bp encoding 251 amino acids, and domain analysis revealed that the predicted amino acids sequence contained two highly conserved SANT domains and binding sites for cold stress factor CBF. By multiple sequence alignment and phylogenetic analysis, it is indicated that AeMYB4 clustered with AtMYB15 from Arabidopsis thaliana, belonging to subgroup S2 of R2 R3-MYB. And most of the transcription factors in this subfamily are related to low temperature stress. The GFP-AeMYB4 fusion protein expression vector for subcellular localization was constructed and transferred into Agrobacterium tumefaciens to infect the leaves of Nicotiana benthamiana, and the results showed the protein was located in the nucleus. To investigate the transcriptional activation, the constructed pGBKT7-AeMYB4 fusion expression vector was transferred into Y2 H Gold yeast cells, which proved that AeMYB4 was a transcription activator with strong transcriptional activity. Real-time quantitative PCR was used to detect the expression of AeMYB4 gene in three different development stages of stolon and in leaves, flowers, and bulbs of A. edulis, which indicated that AeMYB4 transcription factor was tissue-specific in expression, mainly in the stolon development stage, and that the expression was the most active in the middle stage of stolon development, suggesting that AeMYB4 gene may play an important role in stolon development. This study contributes to the further research on the function of AeMYB4 transcription factor in stolon development of A. edulis.
Amino Acid Sequence ; Arabidopsis/metabolism* ; Cloning, Molecular ; Gene Expression Regulation, Plant ; Humans ; Phylogeny ; Plant Proteins/metabolism*