PCR-single Strand Conformation Polymorphism Analysis Research Articles

Short Communication: Identification of Allelic Variation at the Bovine DRA Locus by Polymerase Chain Reaction-Single Strand Conformational Polymorphism

The major histocompatibility complex DRA locus is noteworthy among the major histocompatibility complex class II loci for the little or no variation reported in many species. In cattle, DRA has not been investigated in depth, and the extent of variation at the locus is not yet known. In this study, we used PCR-single strand conformational polymorphism (SSCP) analysis to screen for potential sequence variation in the second exon of bovine DRA, which encodes the antigen-presentation groove. Four unique SSCP patterns were detected among 384 cattle from New Zealand. Sequence analysis revealed that these SSCP patterns represent 4 DRA alleles; and 3 single nucleotide polymorphisms were detected in exon 2. However, all of the single nucleotide polymorphisms were synonymous, and no amino acid change was therefore expected in this region. The polymorphism detected may be linked to variation elsewhere in the gene that affects its structure or function.

Genetic analysis of CC16, OGG1 and GCLC polymorphisms and susceptibility to COPD

The importance of genetic susceptibility in COPD has not been determined. The aim of this study was to investigate the association between susceptibility to COPD and polymorphisms in the Clara cell 16 kDa secretory protein (CC16), 8-hydroxy-guanine glycosylase (OGG1) and glutamatecysteine ligase catalytic subunit (GCLC) genes in a southern Chinese population of Han nationality. A case-control study was performed on 166 paired subjects with or without COPD, who were randomly selected from a pool of 310 paired subjects. These subjects were selected from epidemiological survey participants, with matched-pairs being strictly localized in the Guangzhou urban and Shaoguan rural areas. The following polymorphisms were genotyped by PCR-single strand conformation polymorphism analysis: 38 A/G in exon 1 of the CC16 gene, 1245C/G in exon 7 of the OGG1 gene and -129C/T in the GCLC gene. Genotype frequencies and allelic frequencies were analysed. There were no significant differences in the distribution of genotype frequencies for CC16 38 A/G, OGG1 1245C/G or GCLC -129C/T between the COPD and non-COPD subjects. The distribution of the allelic frequencies of these three genes also showed no significant difference between the two groups. The genetic polymorphisms in CC16 38 A/G, OGG1 1245C/G and GCLC -129C/T are not associated with susceptibility to COPD in a southern Chinese population of Han nationality.

Mutational analysis of the HER2 gene in lung tumors from Caucasian patients: Mutations are mainly present in adenocarcinomas with bronchioloalveolar features

Activating mutations in the tyrosine kinase domain of the HER2 gene have recently been reported in lung adenocarcinomas, mainly in East Asian patients. Our study was devised to evaluate the prevalence and nature of HER2 mutations in lung adenocarcinomas from Caucasian patients. The mutational status of the HER2 gene was evaluated in 403 lung adenocarcinomas by PCR-single strand conformation polymorphism analysis and direct sequencing of Exons 19 and 20. We found HER2 mutations in 9 (2.2%) cases. Seven (78%) of the mutations were in frame duplications/insertions at codons 776-779 (YVMA), the other 2 were base substitutions resulting in aminoacid changes. The hotspot mutation at bases 776-779 was previously found to be the most frequent HER2 mutation in Asiatic patients. The distribution of mutations was significantly different between conventional lung adenocarcinomas (CLAs) and lung adenocarcinomas with bronchioloalveolar features (ABAFs). Seven (6.2%) of 113 ABAFs and 2 (0.7%) of 290 CLA were mutated (p = 0.0025). In addition, the frequency of HER2 mutations was slightly higher in females (4.1%) than in males (1.8%) and in never smokers (3.1%) than in smokers (1.9%), but differences were not statistically significant. This series of tumors was also investigated for EGFR and K-ras mutations. EGFR mutations were observed in 43 (10.7%) cases, and K-ras mutations in 110 (27.3%) cases. EGFR, HER2 and K-ras mutations were found to be mutually exclusive events. The presence of HER2 mutations in a subset of patients with lung adenocarcinoma raise hope to treat these patients with HER2 specific kinase inhibitors.

Deregulation of the p16-cyclin D1/cyclin-dependent kinase 4–retinoblastoma pathway involved in the rat bladder carcinogenesis induced by terephthalic acid-calculi

Prolonged cell proliferation in response to irritation by calculi may itself evoke malignant transformation of the urothelium. However, the molecular mechanisms underlying this process are still unknown. The aim of the present study was to investigate cell cycle regulatory mechanisms in bladder carcinogenesis induced by bladder calculi. Six-week-old Wistar rats were consecutively fed a diet containing 5% terephthalic acid (TPA), 5% TPA plus 4% sodium bicarbonate (NaHCO(3)), 4% NaHCO(3), or basal diet for 48 weeks. Animals were killed at weeks 12, 24, and 48. Treatment with 5% TPA caused high incidences of bladder calculi, preneoplastic lesions, and neoplastic lesions. Immunohistochemical examination revealed overexpression of cyclin D1, cyclin-dependent kinase 4 (Cdk4), retinoblastoma (Rb), and proliferating cell nuclear antigen (PCNA) in bladder preneoplastic and neoplastic lesions. In contrast, p16 expression was reduced or absent. These results were confirmed by immunoblotting analysis. Quantitation of mRNA by real-time reverse transcription-polymerase chain reaction (RT-PCR) showed a significant increase in cyclin D1 and PCNA mRNA in tumor cells. None of the 16 transitional cell carcinomas (TCCs) had ras mutations as examined by PCR-single strand conformational polymorphism (PCR-SSCP) analysis. These results suggested that deregulation of p16-cyclin D1/Cdk4-Rb pathway, but not oncogenic activation of ras, plays a crucial role in bladder tumorigenesis induced by bladder calculi.

Sequence Identification, Tissue Distribution, Mapping and Polymorphism of the Porcine Sar1b Gene

The predicted full-length cDNA sequence of the porcine Sar1b gene was characterized by assembling pig ESTs from GenBank. The coding sequence (CDS) shares high sequence identity with the corresponding sequences of human (93%) and mouse (91%). The reverse transcription-polymerase chain reaction (RT-PCR) displayed that porcine Sar1b gene is expressed in all eight tissues (liver, small intestine, stomach, heart, lung, spleen, muscle and fat). Analysis of the somatic cell hybrid panel (SCHP) and the INRA-University of Minnesota porcine radiation hybrid (IMpRH) panel indicated the gene maps to SSC2 (1/2 q24)-q29 and most closely links to the interleukin-4 (IL4) gene. One base-pair deletion polymorphism in the 3′ untranslated region (UTR) detected by PCR-single strand conformational polymorphism (PCR-SSCP) analysis shows allele frequency differences between Meishan, Yushan Black, Dahuabai, Qingping, Tibetan, Landrace, Large White and Duroc. The association analysis using pigs of Tongcheng, Landrace × (Large White × Tongcheng) and Large White × (Landrace × Tongcheng) suggested that the deletion polymorphism was associated with the porcine muscle pH value.

Assessing the Risk of Biological Control Agents on the Indigenous Microbial Communities: Serratia plymuthica HRO-C48 and Streptomyces sp. HRO-71 as Model Bacteria

The phytopathogenic fungus Verticillium dahliae Kleb. causes high yield losses in strawberry production. As effective chemical control of this fungus is no longer available, biological control based on natural antagonists might provide new control strategies. The aim of this study was to assess the impact of the two biological control agents S. plymuthica HRO-C48 and Streptomyces sp. HRO-71 on the rhizosphere community of the Verticillium host plant strawberry in field trials at two different sites in Germany. Therefore, we determined the abundances of culturable bacteria and investigated the community structure of the total rhizosphere microbiota by PCR-single strand conformation polymorphism analysis of the 16S rRNA and fungal ITS1 region. The abundances of culturable rhizobacteria on R2A medium as well as the proportion of in vitro Verticillium antagonists did not differ significantly. Additionally, no treatment specific differences were obtained in the composition of species of the non-target antagonistic bacteria in the rhizospheres. The culture-independent analysis revealed only transient differences between the bacterial communities not due to the treatments rather than to the plant growth stage. Fungal and bacterial community fingerprints showed the development of a microbiota, specific for a field site. However, no sustainable impact of the bacterial treatments on the indigenous microbial communities was found using culture-dependent and -independent methods.

Survivin promoter polymorphism and cervical carcinogenesis

Background: Survivin, a novel member of the inhibitor of apoptosis family, plays an important role in cell cycle regulation. A common polymorphism at the survivin gene promoter (G/C at position...

Beta-catenin mutations correlate with over expression of C-myc and cyclin D1 Genes in bladder cancer.

We hypothesized that over expression of c-myc and cyclin D1 genes is transcriptionally activated by beta-catenin mutation independent of gene amplification in bladder cancer. To test this hypothesis we investigated the relationship of beta-catenin mutation to c-myc and cyclin D1 mRNA with special reference to the changes in copy number of the 2 genes. Genomic DNA and total RNA were extracted from 59 bladder cancer specimens and from 31 histologically normal specimens of bladder mucosa. We performed beta-catenin deletion screening by polymerase chain reaction (PCR) using primers spanning exons 3 (including the glycogen synthase kinase-3beta consensus motif), 5 and 6. Mutational changes in beta-catenin in exons 3, 5 and 6 were detected by each PCR-single strand conformational polymorphism analysis followed by direct DNA sequencing. mRNA expression and copy numbers of c-myc and cyclin D1 were determined by semiquantitative reverse transcriptase-PCR and competitive genomic PCR. Missense mutations of beta-catenin found in grade 3 bladder cancer were involved in the consensus motif of glycogen synthase kinase-3beta in exon 3. These cancers showed strong intracellular accumulation of beta-catenin and intense expression of c-myc and cyclin D1 mRNA compared with samples lacking the beta-catenin mutation. When grade 3 cancers were compared, expression levels of c-myc and cyclin D1 mRNA were still higher in those with versus without the beta-catenin mutation. In bladder cancers with beta-catenin mutations copy numbers of the c-myc and cyclin D1 genes did not amplify. Bladder cancer harboring a beta-catenin mutation may represent aggressive biological behavior with enhanced proliferating activity. These findings are important for understanding the role of beta-catenin mutation in the pathogenesis of bladder cancer.

Androgen receptor mutants detected in recurrent prostate cancer exhibit diverse functional characteristics

Alterations in the function of androgen receptor (AR) and its signaling pathway may be responsible for the progression of prostate cancer. The goal of the present study was to investigate the potential roles of AR structural and functional alterations in the progression of prostate cancer, and the relationship between the structure and function of the AR. AR gene in 58 prostate cancer samples was examined for mutations using PCR-single strand conformation polymorphism (SSCP) analysis and DNA sequencing. Effects of mutations on the structure and function of AR were investigated by androgen-binding assays and transactivation assays, respectively. Four novel somatic mutations (G142V, D221H, E872Q, and M886I) were identified from recurrent prostate cancer samples. None of the AR mutants differed from wild-type AR (wtAR) in their abilities to bind the synthetic androgen methyltrienolone. However, these mutated AR exhibited diverse functional characteristics as compared with wtAR. G142V and D221H showed increased responses to DHT. E872Q could be abnormally activated by 17beta-estradiol, progesterone, and cyproterone acetate (CPA). Furthermore, E872Q and M886I presented increased responses to DHT in the presence of coactivators TIF-2 and CBP, but not p300. On the other hand, although overexpression of corepressors N-CoR and SMRT could result in evident inhibition on DHT- or CPA-induced transactivity of wtAR and the AR mutants, N-CoR displayed stronger inhibitory effects on DHT-induced transactivity of the AR mutants (especially for E872Q and M886I) than that of wtAR. To our knowledge, this is the first characterization of enhanced inhibitory effects of corepressors on the transactivity of the AR mutants found in prostate cancer. The data presented here demonstrate that AR mutants found in prostate cancer had different functional alterations, which might play an important role in the progression of prostate cancer.

A Patient with TP53 Germline Mutation Developed Bowen's Disease and Myelodysplastic Syndrome with Myelofibrosis after Chemotherapy against Ovarian Cancer

Here we report a case of myelodysplastic syndrome (MDS) with myelofibrosis associated with Bowen's disease. A female patient had undergone an operation and chemotherapy for ovarian cancer when she was 65 years old, and she developed MDS at the age of 70 years old. PCR-single strand conformation polymorphism (SSCP) analysis of peripheral blood mononuclear cells, a Bowen's disease lesion, and normal skin showed an abnormal peak in TP53 exon5. Direct sequencing revealed that they all had missense mutation in codon 175 (G to A) of arginine switched to histidine, suggesting a germline mutation of TP53. It was speculated that p53 function was lost by TP53 germline mutation with the loss of a wild type allele induced by the chemotherapy against ovarian cancer, leading to the development of MDS. No therapeutic effects of low dose melphalan or cyclosporine A on MDS were observed, however one month of 30 mg/day prednisolone administration induced a hematological response.

Lack of association between sigma receptor gene variants and schizophrenia.

Several pharmacological studies suggest the possible involvement of sigma(1) receptors in the pathogenesis of schizophrenia. An association has been reported between schizophrenia and two variants (GC-241-240TT and Gln2Pro) in the sigma(1) receptor gene (SIGMAR1). We also previously reported that, along with T-485 A, these two variants alter SIGMAR1 function. To investigate the role of SIGMAR1 in conveying susceptibility to schizophrenia, we performed a case-control study. We initially screened for polymorphisms in the SIGMAR1 coding region using PCR-single strand conformation polymorphism analysis. The distribution of SIGMAR1 polymorphisms was analyzed in 100 schizophrenic and 104 control subjects. A novel G620A variant was detected in exon4. G620A was predicted to alter the amino acid represented by codon 211 from arginine to glutamine. Our case-control study showed no significant association between the T-485 A, GC-241-240TT, Gln2Pro, and G620A (Arg211Gln) variants and schizophrenia and clinical characteristics. These findings suggest that these SIGMAR1 variants may not affect susceptibility to schizophrenia.

Beta-catenin gene alteration in glandular stomach adenocarcinomas in N-methyl-N-nitrosourea-treated and Helicobacter pylori-infected Mongolian gerbils.

The goal of this study was to elucidate whether beta-catenin gene mutations might contribute to glandular stomach carcinogenesis in Helicobacter pylori (H.pylori)-infected Mongolian gerbils. Firstly, exon 3 of gerbil beta-catenin cDNA, a mutation hot spot, was cloned and sequenced and found to have 89.3% homology with the human form and 95.5% with the rat and mouse forms. Peptide sequence in this region was shown to be 100% conserved in these mammals. Then, 45 stomach adenocarcinomas induced with N-methyl-N-nitrosourea (MNU) plus H. pylori infection and 7 induced with MNU alone were examined for beta-catenin expression by immunohistochemistry and for DNA mutations using a combination of microdissection and PCR-single strand conformation polymorphism analysis. One gastric cancer in the MNU + H. pylori group (2.2%) displayed nuclear (N) beta-catenin localization, 3 (6.7%) showed cytoplasmic (C) distribution in local regions, and 41 (91.1%) demonstrated cell membrane (M) localization. Tumors induced by MNU alone showed only membranous beta-catenin localization (7/7). Analysis of exon 3 of the beta-catenin gene dem-onstrated all tumors with membrane or cytoplasmic staining as well as surrounding normal mucosa (S) to feature wild-type beta-catenin. In contrast, the lesion with nuclear staining had a missense mutation at codon 34 [GAC (Gly) --> GAA (Glu)] in exon 3 (1/1 = 100%, N vs. M, P < 0.05; and N vs. S, P < 0.05). In conclusion, these results suggest that beta-catenin may not be a frequent target for mutation in stomach carcinogenesis in MNU + H. pylori-treated gerbils.

Genetic detection of Chinese hereditary nonpolyposis colorectal cancer.

To explore the germline mutations of the two main DNA mismatch repair genes (hMSH2 and hMLH1) between patients with hereditary non-polyposis colorectal cancer (HNPCC) and suspected (atypical) HNPCC. Genomic DNA was extracted from the peripheral blood of the index patient of each family, and germline mutations of hMSH2 and hMLH1 genes were detected by PCR-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing techniques. For PCR-SSCP analysis, 67% (4/6) abnormal exons mobility in typical group and 33% (2/6) abnormal exons mobility in atypical group were recognized. In direct DNA sequencing, 50% (3/6) mutation of MMR genes in typical group and 33% (2/6) mutation of MMR genes in atypical group were found, and 4/6 (66.67%) and 1/6 (16.67%) mutations of hMSH2 and hMLH1 were identified in typical HNPCC and atypical HNPCC, respectively. Mutation detection of the patients is of benefit to the analysis of HNPCC and, PCR-SSCP is an effective strategy to detect the mutations of HNPCC equivalent to direct DNA sequence. It seems that there exist more complicated genetic alterations in Chinese HNPCC patients than in Western countries.

High susceptibility of nullizygous p53 knockout mice to colorectal tumor induction by 1,2-dimethylhydrazine.

The susceptibility of male p53 nullizygote (-/-), heterozygote (+/-), and wild-type (+/+) mice to 1,2-dimethylhydrazine (DMH) induction of colon carcinogenesis was investigated. In a preliminary short-term experiment, male mice of three genotypes were given s.c. of 20 mg/kg DMH once weekly for 5 weeks. In a medium-term experiment, mice were given weekly s.c. of DMH for 15 weeks. In a long-term experiment, male p53 (+/-) and (+/+) mice were given weekly injections of DMH for 15 weeks, and killed at week 30. In the medium-term experiment, carcinomas were observed in 70% of p53 (-/-) mice, although there were no carcinomas in p53 (+/+) and (+/-) mice. In the long-term experiment, there was no significant difference in incidences of adenomas and carcinomas between p53 (+/+) and (+/-) mice. PCR-single strand conformation polymorphism analysis of exons 5-8 of p53 gene revealed four mutations in one focal atypia, one adenoma, and two carcinomas, out of 56 colonic proliferative lesions in the medium- and long-term experiments. These results suggest that p53 might not be a direct target of DMH but complete loss of p53 might elevate susceptibility to DMH-induced colorectal carcinogenesis.

Distribution of MN genotypes detected by PCR-SSCP analysis

Genotyping of the MN blood system was performed by means of PCR-single strand conformation polymorphism (PCR-SSCP) analysis. Twelve band patterns corresponding to each MN genotype composed of alleles M G, M T, N 1, N 2 and N V were detected. In general, M G or N 1>M T>N 2 in order of decreasing frequency was observed as four common alleles in five Japanese, two Chinese and a German populations. PCR-SSCP analysis provides more discriminative classification to the MN genotyping.

β-Catenin Mutations Correlate with Over Expression of C-myc and Cyclin D1 Genes in Bladder Cancer

We hypothesized that over expression of c-myc and cyclin D1 genes is transcriptionally activated by beta-catenin mutation independent of gene amplification in bladder cancer. To test this hypothesis we investigated the relationship of beta-catenin mutation to c-myc and cyclin D1 mRNA with special reference to the changes in copy number of the 2 genes. Genomic DNA and total RNA were extracted from 59 bladder cancer specimens and from 31 histologically normal specimens of bladder mucosa. We performed beta-catenin deletion screening by polymerase chain reaction (PCR) using primers spanning exons 3 (including the glycogen synthase kinase-3beta consensus motif), 5 and 6. Mutational changes in beta-catenin in exons 3, 5 and 6 were detected by each PCR-single strand conformational polymorphism analysis followed by direct DNA sequencing. mRNA expression and copy numbers of c-myc and cyclin D1 were determined by semiquantitative reverse transcriptase-PCR and competitive genomic PCR. Missense mutations of beta-catenin found in grade 3 bladder cancer were involved in the consensus motif of glycogen synthase kinase-3beta in exon 3. These cancers showed strong intracellular accumulation of beta-catenin and intense expression of c-myc and cyclin D1 mRNA compared with samples lacking the beta-catenin mutation. When grade 3 cancers were compared, expression levels of c-myc and cyclin D1 mRNA were still higher in those with versus without the beta-catenin mutation. In bladder cancers with beta-catenin mutations copy numbers of the c-myc and cyclin D1 genes did not amplify. Bladder cancer harboring a beta-catenin mutation may represent aggressive biological behavior with enhanced proliferating activity. These findings are important for understanding the role of beta-catenin mutation in the pathogenesis of bladder cancer.

Resistance to multiple novel antifolates is mediated via defective drug transport resulting from clustered mutations in the reduced folate carrier gene in human leukaemia cell lines.

We have studied the molecular basis of resistance of multiple human leukaemia CCRF-CEM sublines to the novel antifolates ZD9331, GW1843, AG2034, PT523 and edatrexate, which use the reduced folate carrier (RFC) as their main cellular uptake route and that target different folate-dependent enzymes. Antifolate-resistant sublines established by stepwise and single-step selections displayed up to 2135-fold resistance to the selection drug, and up to 2323-fold cross-resistance to various hydrophilic antifolates. In contrast, these sublines were up to 17- and 20-fold hypersensitive to the lipophilic antifolates AG377 and trimetrexate, respectively. The total reduced folate pool of these antifolate-resistant sublines shrunk by 87-96%, resulting in up to 42-fold increased folic acid growth requirement. These sublines lost 92-97% of parental [(3)H]methotrexate influx rates. Genomic PCR single-strand conformational polymorphism analysis and sequencing revealed that most of these drug-resistant sublines harboured RFC mutations that surprisingly clustered in two confined regions in exons 2 and 3. The majority of these mutations resulted in frame-shift and/or premature translation termination and lack of RFC protein expression. The remaining mutations involved single amino acid substitutions predominantly residing in the first transmembrane domain (TMD1). Some RFC-inactivating mutations emerged during the early stages of antifolate selection and were stably retained during further drug selection. Furthermore, some sublines displayed a markedly decreased or abolished RFC mRNA and/or protein expression. This constitutes the first demonstration of clustering of multiple human RFC mutations in TMD1, thereby suggesting that it plays a functional role in folate/antifolate binding and/or translocation. This is the first molecular characterization of human RFC-associated modalities of resistance to various novel antifolates in multiple leukaemia sublines.

Elevated susceptibility of the p53 knockout mouse esophagus to methyl-N-amylnitrosamine carcinogenesis.

Mutations of the p53 tumor suppressor gene constitute one of the most frequent molecular changes in a wide variety of human cancers, including those in the esophagus. Mice deficient in p53 have recently attracted attention for their potential to identify chemical genotoxins. In this study we investigated the susceptibility of p53 nullizygous (-/-), heterozygous (+/-) and wild-type (+/+) mice to methyl-N-amylnitrosamine (MNAN), which specifically induces esophageal tumors in mice and rats. The p53 (+/-) and (+/+) mice were treated with 5 or 15 p.p.m. MNAN in their drinking water for 8 weeks then maintained without further treatment for an additional 7 or 17 weeks, being killed at experimental weeks 15 or 25. An additional group of p53 (-/-) mice were given 5 p.p.m. MNAN for 8 weeks and killed at week 15. At 15 weeks in the 5 p.p.m. groups, squamous cell carcinomas (SCCs) were observed in 10/12 (83.3%) p53 (-/-) and 1/15 (6.7%) p53 (+/-) mice, but in none of the p53 (+/+) mice. In the animals receiving 15 p.p.m., 2/14 (14.3%) p53 (+/-) and 1/11 (9.1%) p53 (+/+) mice developed SCCs. At 25 weeks, the incidence of SCCs was 7/16 (43.8%) and 8/14 (57.1%) in p53 (+/-) mice and 1/13 (7.7%) and 2/10 (20.0%) in p53 (+/+) mice at 5 and 15 p.p.m., respectively. Of the SCCs examined by PCR-single strand conformation polymorphism analysis, 61% (14/23) from p53 (+/-) and 50% (6/12) from p53 (+/+) mice demonstrated mutations in the p53 gene (exons 5-8). These results indicate the order of susceptibility to MNAN-induced esophageal tumorigenesis to be as follows: nullizygotes (-/-) > heterozygotes (+/-) > wild-type (+/+), and provide strong evidence of involvement of p53 mutations in the development of esophageal SCCs.

Characterization of Salmonella serovars by PCR-single-strand conformation polymorphism analysis.

PCR-restriction fragment length polymorphism (PCR-RFLP) and PCR-single-strand conformation polymorphism (PCR-SSCP) analyses were carried out on the 1.6-kb groEL gene from 41 strains of 10 different Salmonella serovars. Three HaeIII RFLP profiles were recognized, but no discrimination between the serovars could be achieved by this technique. However, PCR-SSCP analysis of the groEL genes of various Salmonella serovars produced 14 SSCP profiles, indicating the potential of this technique to differentiate different Salmonella serovars (interserovar differentiation). Moreover, PCR-SSCP could differentiate strains within a subset of serovars (intraserovar discrimination), as three SSCP profiles were produced for the 11 Salmonella enterica serovar Enteritidis strains, and two SSCP profiles were generated for the 7 S. enterica serovar Infantis and five S. enterica serovar Newport strains. PCR-SSCP has the potential to complement classical typing methods such as serotyping and phage typing for the typing of Salmonella serovars due to its rapidity, simplicity, and typeability.

Identification of Th2-type suppressor T cells among in vivo expanded ocular T cells in mice with experimental autoimmune uveoretinitis.

Experimental autoimmune uveoretinitis (EAU), which is a T cell mediated organ specific autoimmune disease, is induced by immunization with interphotoreceptor retinoid binding protein (IRBP) in susceptible strains of mice. It has been found that IRBP-derived peptide 518-529 (p518-529) generates Th2-type responses and inhibits IRBP-induced EAU, indicating that the p518-529 might be an epitope for suppressor T cells in IRBP-induced EAU. First, we observed that there were T cells producing the Th2 type cytokines such as IL-4 and IL-10 in late phase of EAU. Furthermore, to examine whether p518-529-reactive T cells expand in the eye during EAU, T cell receptor (TCR) of ocular T cells was compared with that of p518-529 reactive T cells in spleen from mice with EAU by PCR-single strand conformation polymorphism (PCR-SSCP) and nucleotide sequence analysis. SSCP and sequence analyses indicated that p518-529 reactive TCR BV10+ T cells bearing amino acid motif(PWG) and TCR BV13+ T cells bearing amino acid motif(PGLGGY) in their complementary-determining region 3 (CDR3) region were clonally expanding in ocular tissues on day 28 after immunization, although these T cells were not detected on day 14. These findings demonstrate that p518-529 reactive Th2-type T cells expand oligoclonally in the uveitic eyes in the late stage of EAU and may function as Th2-type suppressor T cells for improvement of the disease.