COMPARISON OF DIFFERENT STAINING METHODS FOR ANALYSING ESTERASE (EST) ISOZYME OF FUNGI AND PLANTS

EST isozymes are one of the frequently used biochemical markers in genetic analysis of fungi. and the staining is an important process in electrophoresis analysis of studying Est.The effects were compared ainong different stain recipes for Est of 3 kinds of fungi-Lentinus edodes. Pleurotus sapidus. Phellinus igriarius and 2 kinds of plants-Populus sp and Brassica chinensis. Of the four kinds of Est staining recipes tested.the recipe α-acetic acid-naphther showed the best effect.and followed by β-aceticacid-naphther, semicontent α-aceticacid-naphther and α+β-aceticacidnathpher.

A Comparative Study on Four Staining Methods for Antitumor Active Fraction of Periplaneta americana

[ Objective ] This study aimed to compare four staining methods for proteins of SDS-polyacrylamide gel dectrophoresis （SDS-PAGE） and explore a suit- able staining method for the antitumor active fraction of P. americana after polyacrylamide gel electrophoresis. [ Method ] BSA was used as the standard for the comparison of Coomassie brilliant blue staining method, potassium staining method, calcium staining method and silver staining method, on the basis, antitumor ac- tive fraction samples of P. americana were used for SDS-PAGE electrophoresis and staining. [ Result] The results showed that silver staining method could be ac- curately, quickly and easily used for SDS-PAGE staining of the antitumor active fraction of P. amer/cana. [ Conclusion] This study laid the foundation for explo- ring the medicinal value of P. americana.

Sensitivity and specificity of different staining methods to monitor apoptosis induced by oxidative stress in adherent cells

Objective To study the sensitivity and specificity of different staining methods to monitor apoptosis induced by oxidative stress in adherent cells.Methods Sensitivity and specificity of several common methods for apoptosis determination were evaluated (Apo2.7-expression, Annexin V-binding, TUNEL-reaction, poly-(ADP-ribose)-polymerase-(PARP) cleavage and single-stranded-DNA (ssDNA) staining). Apoptosis was induced by oxidative stress generated by hydrogen peroxide in 3 cultured cells types growing as adherent monolayer (MiaPaCa-2, Hep-G2 and human skin fibroblasts), necrosis was induced by depletion of cellular ATP using sodium azide. Cells positively stained by the respective apoptosis assay were quantified and alterations of cell morphology were monitored by fluorescence microscopy. The date was analyzed by one-way analysis of variance and significance test of correlation coefficient.Results One hour after apoptosis induction significant cell fractions were positively stained for ssDNA (33% with MiaPaCa-2 cells, 35% with Hep-G2 cells, 56% with human skin fibroblasts). PARP-cleavage was less sensitive compared to the ssDNA-staining. Apo2.7-expression, Annexin V-binding and TUNEL-reaction were not applicable to detect early apoptosis induced by oxidative stress (below 2 hours), but were efficiently monitoring late apoptosis. Specificity of ssDNA-staining was complete with each cell type even 4 hs after induction of necrosis by the highest sodium azide concentration. In contrast, the same experimental conditions resulted in 50%-90% positively stained necrotic cells by using Apo2.7-expression, TUNEL-reaction or AnnexinV-binding. Surprisingly, specificity of PARP-cleavage was highly depending on the respective cell type.Conclusions Our study prove that among the five methods investigated only ssDNA-staining allowed to completely differentiate apoptosis from necrosis, and is thus suitable to reliably detect early as well as late apoptosis. Therefore, the ssDNA-staining may be used as reference method to clearly identify apoptosis induced by oxidative stress in adherent cells. The TUNEL-reaction, annexin-V-binding and Apo-2.7-expression may be used to quantify the number of apoptotic and necrotic cells especially at later stages but without discrimination of apoptosis and primary or secondary necrosis.

Two New Identification Methods for Encephalitozoon cuniculi on Tissue Section

[ Objective] The paper aimed to search new identification methods of Encephalitozoon cuniculi on tissue sections. [ Method] Using improved Gram staining method and methyl green pyronin staining method, the pathological sections of sick rabbits were stained and identified. [ Result] The pathological changes in brain tissue could be clearly observed on sections, but parasites were not examined in pathological brain tissues stained by common staining method. When the pathological section was stained by improved Gram staining method, the pathological changes in brain tissue were not ouly stained very clearly, but blue parasites were also found in brain tissues. The parasites in epithelioid cells were stained into purple ones by methyl green pyronin staining method. [ Conclusion] The im- proved Gram staining method and methyl green pyronin staining method performed good staining effects of E. cuniculi in pathological sections, which were conducive to rapid diagnosis of encephalitozoonosis in rabbit.

A RAPID STAINING METHOD SHOWING AgNOR AND DNA IN PATHOLOGICAL DIAGNOSIS

In order to satisfy the need of rapid clinical diagnosis, a series of experimental studies for AgNOR (Argyrophilic NORassociated proteins) and DNA (Dereyribonucleic acid) staining was compared, from which a stable staining solution was selected to show mucin residence. Using the referred tables, with reference to the corresponding relation of the staining solution with temperature and time, reliable staining results for AgNOR and DNA could be obtained.These two modified staining methods are simple, reliable and easy to operate, able to provide good contrast for pathological diagnosis.

A NEW STAINING METHOD FOR THE MEASUREMENT OF PROTEIN USING TETRAPHENYLPORPHYRIN TETRASULFONATE(TPPS_4)

A new dye-staining method for protein assay is described.The reaction between TPPS_4 and protein molecule causes a shift in the absorption of TPPS_4 from 435 nm to 488nm.The absorbance at 488 nm is proportional to the concentration of protein.The range of Beer's law was 1-5 ug/ml and the Sandell's sensitivity was 0.0087 ug/cm^2.

Induced Differentiation of Epithelioid Carcinoma Cell Lines: Evidence for Tumor Cell Quantal Mitosis

The effects of growth factors and calcium concentrations present in different culture media on induction of terminal differentiation were investigated for four different epidermoid carcinoma cell lines, Hela, KB, A431, and SCC-25, and their responses determined relative to those elicited by normal human keratinocytes subjected to these culture conditions. Differentiation status was determined cyto-chemically by a validated keratin protein staining method, and by autoradiographic analyses. Growth and differentiation promoting factors that influenced the direction of integrated control of growth and differentiation in normal human keratinocytes were found to be effective for some cell lines but not others. The factors examined were 1) high density arrest in serum-free and serum-containing media, 2) media shifts from high density culture in serum-containing media to low density growth factor-depleted or supplemented serum-free medium, and 3) the concentration of calcium in the media. The extent and degree of differentiation achieved varied among different cell lines depend on the presence or absence of serum, EGF and insulin protein growth factors. Certain growth media appear to sponsor keratin protein, cyto-chemically-detected differentiation, and evidence of quantal mitotic division in low density HeLa cell and SCC25 cell cultures. Epidermoid carcinoma cell lines retain limited capacity to commit to early stages of cell differentiation.

Expression and clinical significance of HIF-1α,VEGF and Survivin in esophageal squamous cell carcinoma

Objective： To evaluate the expression and correlation of hypoxia inducible factor la （HIF-la） and vascular endothelial growth factor （VEGF） and Survivin proteins in biopsy specimens of esophageal squamous cell carcinoma （ESCC）, and then determine whether the levels of expression of these proteins could predict the clinical effectiveness of radiotherapy in individual cancers. Methods： The expressions of HIF-IQ, VEGF and Survivin were shown by S-P immunohistochemical staining method in biopsy specimens of ESCC, which were obtained endoscopically from 50 patients before radiotherapy, and 10 cases of normal esophageal tissue. Results： The positive expression rates of H IF-IQ, VEGF and Survivin were 68%, 74% and 72% in ESCC respectively. However, the three tumor markers had negative expressions in normal esophageal tissue. The positive rate of HIF-IQ was positively correlated with VEGF and Survivin proteins. The positive rates of HIF-IQ and Survivin were closely related to the clinical stage, radiotherapy effectiveness and survival, otherwise, the expression of HIF-IQ was closely related to distant metastasis; both of them were no correlation with the differentiation degree of tumor. The effective rates of radiotherapy and mean survival periods of those cases with positive and negative expressions of HIF-IQ were 8.8%, 10 months and 81.25%, 25 months, respectively. The one, two, and three years survival rates of patients with positive and negative expressions of HIF-IQ were 38.2%, 5.9%, 2.9%, and 81.3%, 54.2%, 15.8%, respectively （P = 0.001）. Patients with HIF-IQ positive expression obviously survived less than those with negative expression and the difference was significant. The expression of VEGF was only related to the distant metastasis. Conclusion： Over expressions of HIF-IQ, VEGF and Survivin were found in ESCC. The positive rate of HIF-IQ was positively correlated with VEGF and Survivin proteins. The expression of HIF-IQ may serve as an important parameter in evaluating response for radiotherapy and prognosis of ESCC. It may play an important role by up-regulating the transcription of VEGF and Survivin genes.

Expression of IL-34 in Gastric Adenocarcinoma Tissues and Its Relationship with Apoptosis

[Objectives]To explore the expression of interleukin 34(IL-34)in gastric adenocarcinoma tissues and its relationship with apoptosis of gastric adenocarcinoma cells.[Methods]60 cases of surgically resected human gastric adenocarcinoma tissue specimen and 50 cases of adjacent normal gastric mucosa tissue specimen were collected,and the expression of IL-34 protein was determined by immunohistochemical streptavidin-perosidase(SP)method.Cell apoptosis in tissue specimen was detected by TUNEL staining method,and the relationship between IL-34 protein expression and apoptosis of gastric adenocarcinoma cells was analyzed.[Results]Immunohistochemical SP experiment indicated that the expression of IL-34 protein in gastric adenocarcinoma was higher than that in adjacent normal gastric mucosa(P<0.05);its positive expression was related to histological differentiation,TNM stage,invasion depth,and lymph node metastasis of gastric adenocarcinoma(P<0.05),but not to gender and age(P>0.05).TUNEL experiment showed that compared with the adjacent normal gastric mucosa group,the apoptosis index(AI)of gastric adenocarcinoma group was significantly lower(P<0.05);the AI was related to histological differentiation,TNM stage,tumor invasion depth and lymph node metastasis of gastric adenocarcinoma(P<0.05),but not to gender and age(P>0.05).In gastric adenocarcinoma,the AI of IL-34 positive expression group was lower than that of IL-34 negative expression group,and the results were statistically significant(P<0.05).[Conclusions]IL-34 has high expression in gastric adenocarcinoma tissues and is negatively correlated with cancer cell apoptosis.Abnormal expression of IL-34 is involved in the occurrence and development of gastric adenocarcinoma.This provides a new idea for the pathogenesis research and clinical treatment of gastric adenocarcinoma.