Prediction of T cell and B cell epitopes of the 22-, 47-, 56-, and 58-kDa proteins of Orientia tsutsugamushi

Objective: To predict B cell and T cell epitopes of 22-kDa, 47-kDa, 56-kDa and 58-kDa proteins. Methods: The sequences of 22-kDa, 47-kDa, 56-kDa and 58-kDa proteins which were derived from Orientia tsutsugamushi were analyzed by SOPMA, DNAstar, Bcepred, ABCpred, NetMHC, NetMHC II and IEDB. The 58-kDa tertiary structure model was built by MODELLER9.17. Results: The 22-kDa B-cell epitopes were located at positions 194-200, 20-26 and 143-154, whereas the T-cell epitopes were located at positions 154-174, 95-107, 17-25 and 57-65. The 47-kDa protein B-cell epitopes were at positions 413-434, 150-161 and 283-322, whereas the T-cell epitopes were located at positions 129-147, 259-267, 412-420 and 80-88. The 56-kDa protein B-cell epitopes were at positions 167-173, 410-419 and 101-108, whereas the T-cell epitopes were located at positions 88-104, 429-439, 232-240 and 194-202. The 58-kDa protein B-cell epitopes were at positions 312-317, 540-548 and 35-55, whereas the T-cell epitopes were located at positions 415-434, 66-84 and 214-230. Conclusions: We identified candidate epitopes of 22-kDa, 47-kDa, 56-kDa and 58-kDa proteins from Orientia tsutsugamushi. In the case of 58-kDa, the dominant antigen is displayed on tertiary structure by homology modeling. Our findings will help target additional recombinant antigens with strong specificity, high sensitivity, and stable expression and will aid in their isolation and purification.

Prediction of T cell and B cell epitopes of the 22-, 47-, 56-, and 58-kDa proteins of Orientia tsutsugamushi

Objective: To predict B cell and T cell epitopes of 22-kDa, 47-kDa, 56-kDa and 58-kDa proteins. Methods: The sequences of 22-kDa, 47-kDa, 56-kDa and 58-kDa proteins which were derived from Orientia tsutsugamushi were analyzed by SOPMA, DNAstar, Bcepred, ABCpred, NetMHC, NetMHCⅡ and IEDB. The 58-kDa tertiary structure model was built by MODELLER9.17. Results: The 22-kDa B-cell epitopes were located at positions 194-200, 20-26 and 143-154, whereas the T-cell epitopes were located at positions 154-174, 95-107, 17-25 and 57-65. The 47-kDa protein B-cell epitopes were at positions 413-434, 150-161 and 283-322, whereas the T-cell epitopes were located at positions 129-147, 259-267, 412-420 and 80-88. The 56-kDa protein B-cell epitopes were at positions 167-173, 410-419 and 101-108, whereas the T-cell epitopes were located at positions 88-104, 429-439, 232-240 and 194-202. The 58-kDa protein B-cell epitopes were at positions 312-317, 540-548 and 35-55, whereas the T-cell epitopes were located at positions 415-434, 66-84 and 214-230. Conclusions: We identified candidate epitopes of 22-kDa, 47-kDa, 56-kDa and 58-kDa proteins from Orientia tsutsugamushi. In the case of 58-kDa, the dominant antigen is displayed on tertiary structure by homology modeling. Our findings will help target additional recombinant antigens with strong specificity, high sensitivity, and stable expression and will aid in their isolation and purification.

Protection mechanism of deacetylase inhibitor on spleen of rats with severe hemorrhagic shock

Objective To explore the protection and molecular mechanism of histone deacetylase inhibitors (HDACIs) on the spleen of rats with hemorrhagic shock. Methods A total of 60 SPF male SD rats were selected for the modeling of severe hemorrhagic shock using the method of arterial and venous cannulation with the time-divided bleeding. The measurement of mean arterial blood pressure and blood lactic acid was used to verify the modeling. The modeled rats were randomly divided into shock group, shock + suberoylanilide hydroxamic acid (SAHA) group, shock + autogenous transfusion group and shock + SAHA + autogenous transfusion group. Three hours after the treatment, the spleen of rats was collected and TUNEL method was employed to detect the apoptosis of spleen cells in each group. The statistical analysis was performed. Afterwards, real-time PCR and western blot were employed to detect the expression of BCL-2, BAX and caspass3 in the spleen of rats in each group. Results A total of 53 rats had successful modeling of severe hemorrhagic shock, with success rate of 88%. Cell apoptosis in the severe hemorrhagic model group was the most serious. After the intervention of HDACIs and the autogenous transfusion, the tissue injury was a bit recovered. Cell apoptosis was least in the shock + SAHA + autogenous transfusion group (P < 0.05). After the intervention of HDACIs and the autogenous transfusion, the relative expression of BCL-2 was significantly increased (P < 0.05), with highest relative expression of BCL-2 in shock + SAHA + autogenous transfusion group (P < 0.05). After the intervention of HDACIs and the autogenous transfusion, the relative expression of BAX was significantly decreased (P < 0.05), with lowest relative expression of BAX in the intervention group of single HDACIs. The change in the expression of caspass3 was similar to BAX, namely the relative expression of caspass3 was significantly decreased after the intervention of HDACIs and the autogenous transfusion (P < 0.05). Conclusions HDACIs and autogenous transfusion can all protect the spleen injury because of the severe hemorrhagic shock. Its molecular mechanism may be related to the regulation on the expression of BCL-2/BAX and caspass3, which may affect the apoptosis process of cells.

Protection mechanism of deacetylase inhibitor on spleen of rats with severe hemorrhagic shock

OBJECTIVE@#To explore the protection and molecular mechanism of histone deacetylase inhibitors (HDACIs) on the spleen of rats with hemorrhagic shock.@*METHODS@#A total of 60 SPF male SD rats were selected for the modeling of severe hemorrhagic shock using the method of arterial and venous cannulation with the time-divided bleeding. The measurement of mean arterial blood pressure and blood lactic acid was used to verify the modeling. The modeled rats were randomly divided into shock group, shock + suberoylanilide hydroxamic acid (SAHA) group, shock + autogenous transfusion group and shock + SAHA + autogenous transfusion group. Three hours after the treatment, the spleen of rats was collected and TUNEL method was employed to detect the apoptosis of spleen cells in each group. The statistical analysis was performed. Afterwards, real-time PCR and western blot were employed to detect the expression of BCL-2, BAX and caspass3 in the spleen of rats in each group.@*RESULTS@#A total of 53 rats had successful modeling of severe hemorrhagic shock, with success rate of 88%. Cell apoptosis in the severe hemorrhagic model group was the most serious. After the intervention of HDACIs and the autogenous transfusion, the tissue injury was a bit recovered. Cell apoptosis was least in the shock + SAHA + autogenous transfusion group (P < 0.05). After the intervention of HDACIs and the autogenous transfusion, the relative expression of BCL-2 was significantly increased (P < 0.05), with highest relative expression of BCL-2 in shock + SAHA + autogenous transfusion group (P < 0.05). After the intervention of HDACIs and the autogenous transfusion, the relative expression of BAX was significantly decreased (P < 0.05), with lowest relative expression of BAX in the intervention group of single HDACIs. The change in the expression of caspass3 was similar to BAX, namely the relative expression of caspass3 was significantly decreased after the intervention of HDACIs and the autogenous transfusion (P < 0.05).@*CONCLUSIONS@#HDACIs and autogenous transfusion can all protect the spleen injury because of the severe hemorrhagic shock. Its molecular mechanism may be related to the regulation on the expression of BCL-2/BAX and caspass3, which may affect the apoptosis process of cells.

Effects of traditional Chinese medicine on the differentiation of mesenchymal stem cells to osteoblasts / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the effects of traditional Chinese medicine on the differentiation of mesenchymal stem cells (MSC) to osteoblasts.</p><p><b>METHODS</b>Six male Beagle dogs weighed 10-15 kg each were divided into three groups, group A: medicine serum group, group B: non-medicine serum group and group C: bovine serum group. The serum of group A was obtained from the femoral artery of 2 Beagle dogs drinking equivalent dose of traditional Chinese medicine according to body surface area for 7 continuous days. The serum of group B was collected from the femoral artery of 2 Beagle dogs fed with equal volume of normal saline for 7 days. The serum of group C was fetal bovine serum. The tibia marrow was harvested from another 2 Beagle dogs and MSC were isolated and purified by density gradient centrifugation. MSC were cultured in DMEM solution with fetal bovine serum. After MSC were digested by trypsin, MSC were cultured in DMEM solution with the osteogeneic inducer, which contained dexamethasone, antiscorbutic and beta-glycerophosphate. Morphological and histological changes of the MSC were observed under an inverted microscope. Alizarin monosulfonate and nitric acid argentum staining was performed to observe the calcium deposition. MSC were curtured in DMED solution with medicine serum (group A), non-medicine serum (group B) and bovine serum (group C) respectively. The growth curve was detected by the methyl thiazolyl tetrazolium (MTT). The alkaline phosphatase (ALP) activities were detected to observe the differentiation of MSC.</p><p><b>RESULTS</b>The original MSC were observed as fibroblast-like cell shapes. After the osteogeneic inducer was added, MSC were polygon cells with a few polyprocess. Calcium deposition appeared during 10-14 days and alizarin monosulfonate and Von Kossa staining presented positive. MTT results showed that the number of adherent cells of group A was more than that of group B and that of group C significantly after 6 days (P < 0.05). ALP detection proved that ALP activity of group A was more than that of group B and that of group C significantly after 5 days (P < 0.05).</p><p><b>CONCLUSIONS</b>The traditional Chinese medicine promotes the differentiation of MSC to osteoblasts and osteogenesis.</p>

wt-p53 gene repair for p53 mutations of salivary pleomorphic adenoma cells / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To analyze the original mutated codon of p53 gene of salivary pleomorphic adenoma (SPA) and to evaluate the repair effects of wt-p53 on SPA cells.</p><p><b>METHODS</b>Four cases of SPA were obtained from clinical fresh samples and SPA cells were separated and cultured, and then the cells were transduced by Ad-wt-p53. The cells and the corresponding tumor tissue DNA were extracted, PCR and single strand conformational polymorphism (SSCP) and DNA sequencing analysis were performed.</p><p><b>RESULTS</b>PCR-SSCP analysis showed 3 out of 4 SPA with abnormal exon 8 and abnormal exon 6. DNA sequencing analysis showed that exon 6 point mutation was found at codon 203 (GTG-->GCG), poly-codon mutations were found in exon 8 at codon 272 (GTG-->GT square), 275 (TGT-->T square T), 276 (GCC--> square CC) and at codon 290 (CGC-->CGCC). After transduced by Ad-wt-p53, all of the mutated codons were repaired.</p><p><b>CONCLUSIONS</b>p53 gene mutation involved many codons that occurred frequently in the tumorigenesis of SPA. Exogenous wt-p53 could compensate and repair all the mutated p53 codons of SPA cells. SPA cells transduced by Ad-wt-p53 showed the typical apoptosis.</p>

Study of p16INK4/CDKN2 gene homozygous deletions and point mutation in squamous cell carcinoma of buccal mucosa / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To explore the correlation between homozygous deletions and mutation of p16 gene and the carcinogenesis and progression of squamous cell carcinoma of buccal mucosa.</p><p><b>METHODS</b>Thirty buccal cancers, 10 leukoplakias and 8 buccal mucosas were involved. DNA was extracted from the tissues. PCR was used to analyses homozygous deletion of p16 gene. PCR-SSCP-DNA sequencing was performed to detect the point mutation of p16 gene. Immunohistochemical techniques were used to detect the expression of P16 protein.</p><p><b>RESULTS</b>Gene deletions and point mutations were not found in leukoplakia and normal buccal mucosa. Gene deletions were found in 7 samples out of 30 cases of squamous cell carcinoma of buccal mucosa (23.3%), while point mutations were found in 5 samples out of 30 cases of squamous cell carcinoma of buccal mucosa (16.7%). Sequencing analysis showed that 5 cases point mutations were missense mutations, occurred on exon 2. Three cases occurred in the same point, codon 99 (GAT --> AAT). The result of immunohistochemical stains showed that 11 out of 12 cases gene inactivation did not expressed P16 protein.</p><p><b>CONCLUSION</b>Homozygous deletion and point mutation of p16 were the main pattern of gene inactivation in squamous cell carcinoma of buccal mucosa. There was a closely correlation between p16 gene inactivation and the carcinogenesis of squamous cell carcinoma of buccal mucosa.</p>

The relationship between methylation of p16 INK 4/CDKN2 gene in promoter region and progress of squamous cell carcinoma of buccal mucosa / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To determine the correlation between methylation of p16 gene in promoter region and the carcinogenesis and progression of squamous cell carcinoma (SCC) of buccal mucosa.</p><p><b>METHODS</b>Methylation of pl6 gene in SCC and leukoplakia of buccal mucosa was investigated by MSP and pl6 protein was analyzed by Western blot.</p><p><b>RESULTS</b>The methylation of p16 gene was found in 15 of 30 cases SCC and 1 of 10 cases of leukoplakia of buccal mucosa (P < 0.05). Methylation of p16 gene in SCC of buccal mucosa was not related with age, sex, cell differentiation and clinical stage. But methylation of p16 in the cases with lymph node-metastasis was higher than that in the cases without lymph node-metastasis protein (P < 0.05). Meanwhile Methylation of p16 gene was positively correlated with no-expression of p16 protein (P < 0.01).</p><p><b>CONCLUSIONS</b>The methylation of p16 gene leaded to the inactivation of p16 gene and was related with the carcinogenesis and progress of SCC of buccal mucosa.</p>

The effects of HSV-tk suicide gene and wild-type p53 gene on pleomorphic adenoma cells of salivary gland / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To study the therapeutic effects of combined gene therapy of wild type p53 (wt-p53) and herpes simplex virus thymidine kinase (HSV-tk) gene on pleomorphic adenoma cells of salivary gland.</p><p><b>METHODS</b>Wild type p53 and HSV-tk gene were transfected into human pleomorphic adenoma cells of salivary gland by using recombinant adenovirus vector. The efficiency of transfection was checked and gene was expressed by RT-PCR methods. The cell inhibition after transfected was verified by light microscope and MTT.</p><p><b>RESULTS</b>The proliferation of the pleomorphic adenoma cells transfected wt-p53 and HSV-tk gene was inhibited and the cell survival rate decreased to 54% and 38% respectively in 5 days. However, when wt-p53 gene combined with HSV-tk/GCV system, the killing effects was significantly stronger (P < 0.05) and the cell survival rate decreased to 20%.</p><p><b>CONCLUSION</b>Combining p53 gene with HSV-tk/GCV system for gene therapy in pleomorphic adenoma cells of salivary gland is a valuable method.</p>