T cell receptor (TCR) repertoire and function of human epidermal T cells: restricted TCR Vα-Vβ genes are utilized by T cells residing in the lesional epidermis in fixed drug eruption

Human epidermis contains a phenotypically heterogeneous population of T cells. No information, however, is available regarding the TCR repertoire of these T cells and their relevant physiologic and pathologic functions in vivo. To this end, T cells were prepared from the lesional epidermis in two patients with fixed drug eruption (FDE) and their phenotype, function and TCR repertoire were examined in parallel. Both epidermal T cells, termed FDE-1 and -2 cells, respectively, expressed αβ TCR, but displayed some phenotypic heterogeneity. These T cells were induced to display cytolytic activity by ligation of the CD3/TCR-αβ complex. Comparative analyses of TCR Vα and Vβ expression in the epidermal T cells and the paired peripheral blood lymphocytes (PBL) by quantitative polymerase chain reaction (PCR) demonstrated that the epidermal T cells, but not the paired PBL, utilized a very limited range of Vα and Vβ genes. These results indicate that some expansion or preferential migration of epidermal T cells that recognize a restricted set of antigens expressed within the epidermis could occur in situ following ingestion of the causative drug. The persistence of these epidermal T cells in FDE lesions suggests their pathologic role in a drug-induced flare.     

Microarray analysis of T-2 toxin-induced liver, placenta and fetal liver lesions in pregnant rats

Pregnant rats on day 13 of gestation were treated orally with 2 mg/kg of T-2 toxin and sacrificed at 1, 3, 6, 9 and 12 h after the treatment (HAT). Histopathologically, the number of apoptotic cells was increased in the liver, placenta and fetal liver (peaked at 6, 12 and 9-12 HAT, respectively). To examine the gene expression profiles, we performed microarray analysis of these tissues at two selected time points based on the results of the TdT-mediated dUTP nick end labeling (TUNEL) staining. Increased expression of oxidative stress- and apoptosis-related genes was detected in the liver of dams, placenta and fetal liver of pregnant rats treated with T-2 toxin at the peak time point of apoptosis. Decreased expression of lipid metabolism- and drug-metabolizing enzyme-related genes was also detected in these tissues. The results suggested that the mitogen-activated protein kinase (MAPK) pathway might be involved in the mechanism of T-2 toxin-induced apoptosis. In addition, increased expression of the c-jun gene was consistently observed in these tissues. Our results suggest that the mechanism of T-2 toxin-induced toxicity in pregnant rats is due to oxidative stress followed by the activation of the MAPK pathway, finally inducing apoptosis. The c-jun gene may play an important role in T-2 toxin-induced apoptosis.     

Curcumin inhibits telomerase activity in human cancer cell lines

Curcumin, one of the major components of tumeric, the dried rhizome of Curcuma longa L, has been shown to have anti-proliferating and anti-carcinogenic properties. In this study, we examined the effects of curcumin on cell growth and telomerase activity in human cancer cell lines Bel7402, HL60 and SGC7901. Curcumin (1-32 microM) showed anti-proliferating effects on these cell lines in a dose-dependent manner in vitro, and anti-tumor effects when curcumin (50-200 mg/kg) was orally administered to nude mice transplanted with the cancer cells. When the cells were treated with 1 microM of curcumin for 120 h, apoptotic cells were observed by means of the adridine orange/ethidium bromide staining method, single cell microgel electrophoresis and flow cytometric analysis. On the other hand, suppression of telomerase activity in extracts of the cells treated with 1 microM of curcumin was observed by means of a telomeric repeat amplification protocol - silver staining assay. These results suggest that curcumin could suppress telomerase activity in the cancer cell lines and that the decrease of telomerase expression followed by induction of apoptosis might be involved in the anti-proliferating effect of curcumin.     

Morphological and microarray analysis of T-2 toxin-induced rat fetal brain lesion.

To examine morphological and gene expression changes induced by T-2 toxin in the fetal brain in detail, pregnant rats on day 13 of gestation were treated orally with a single dose of T-2 toxin (2 mg/kg) and sacrificed at 1, 3, 6, 9, 12 and 24 h after treatment (HAT). Histopathologically, the number of apoptotic neuroepithelial cells in the telencephalon increased from 1 HAT and peaked at 12 HAT. Based on the histopathological examinations, microarray analysis was performed at 6, 12 and 24 HAT. Microarray analysis showed that the expression of oxidative stress-related genes (heat shock protein 70 (HSP70) and heme oxygenase (HO)) was strongly induced by T-2 toxin at 12 HAT, the peak time point of apoptosis induction. The expression of mitogen-activated protein kinase (MAPK)-related genes (MEKK1 and c-jun) and other apoptosis-related genes (caspase-2 and insulin-like growth factor-binding protein-3 (IGF-BP3)) was also induced by the T-2 toxin treatment. The changes observed by microarray analysis were confirmed for four up-regulated genes (HSP70, HO, IGF-BP3 and VEGF-A) using real-time RT-PCR. Our results suggest that the T-2 toxin-induced apoptosis in the fetal brain is due to oxidative stress, and that the MAPK pathway may be involved in T-2 toxin-induced toxicity.     

中药三七化学成分的 HPLC/ESI-MS分析

目的建立分析中药三七的HPLC-UV-MS方法, 鉴定三七的化学成分.方法色谱柱: Phenomenex Luna C18 柱 (250 mm×4.6 mm ID, 5 μm); 流动相:0.005%甲酸水溶液(A) 和0.005%甲酸乙腈溶液(B), 梯度洗脱;紫外检测波长范围:195-400 nm; 采用正负离子检测模式.结果三七成分获得了良好的分离与检测.通过与对照品的保留时间、正负离子质谱比较, 鉴定了14个成分, 根据正负离子质谱数据和文献, 分析推断了41个成分, 其中, 野三七皂苷-H、野三七皂苷-E、竹节参皂苷-L5、丙二酰基人参皂苷-Rg1、以及三七皂苷-J、-A、-R1、-G、-R2和人参皂苷-Rh3的异构体为首次在三七中发现.结论本方法灵敏度高、分离度好, 适合三七成分的鉴定.本结果有助于进一步对三七进行活性成分研究及质量控制.     

Genetic mapping of allelic loss on chromosome 6q within heterogeneous prostate carcinoma

A number of genetic events have been reported in prostate carcinogenesis, including frequent loss of heterozygosity (LOH) on chromosomes 8q, 10q, 16q and 18q, In samples of heterogeneous, multifocal prostate carcinomas, we focused on chromosome 6q using PCR-based techniques with 15 microsatellite markers to identify the specific 6q deletion within tumors. LOH of one or more polymorphic markers was detected in 10 of 21 tumors (48%). Two of these 10 tumors demonstrated LOH in all cancerous foci at specific loci and 4 tumors showed deletion in one focus. Different deletion patterns were found in 3 tumors when different polymorphic markers were used. In 90% of tumors showing LOH in one or more foci, however, two common regions of LOH were identified; one at 1.81 cM on 6q15–16.3 between markers D6S1631 and D6S1056, and the other at 5.11 cM on 6q16–21 between markers D6S424 and D6S283. By RT-PCR analysis, the TAK1 gene located at these loci did not correlate with LOH status, indicating that TAK1 is not a target gene in prostate carcinoma. The 6q deletion occurs heterogeneously and LOH was more frequent in tumors of higher pathological stages, implying that this alteration is a late event in prostate carcinogenesis. Because prostate carcinomas are genetically multicentric and of multifocal origin, it remains unclear whether the foci containing 6q deletions specifically expand within tumors or to what extent they contribute to the histological heterogeneity characteristic of the disease.     

Alterations of p14ARF and p16INK4a genes in salivary gland carcinomas

The p14ARF and p16INK4a genes are localized to 9p21, where genetic alterations have been reported to be frequent in various human neoplasms. To elucidate their status in salivary gland tumorigenesis, we analyzed a series of 36 salivary gland carcinomas (SGCs) using methylation-specific PCR, differential PCR and immunohistochemistry. Homozygous deletion (3 cases) or methylation (7 cases) of p14ARF was detected in 10 (28%) SGCs, one and three showing co-deletion and co-methylation of both p14ARF and p16INK4a genes, respectively. A total of 5 (14%) SGCs demonstrated homozygous deletion (1 case) or methylation (4 cases) of p16INK4a, all but one being adenoid cystic carcinomas. Immunohistochemical study revealed loss of p14ARF and p16INK4a expression in 11 samples (31%), correlating with the gene status. These results indicate that inactivation of p14ARF and p16INK4a genes by either homozygous deletion or promoter hypermethylation may be important for the molecular pathogenesis of salivary malignant tumors, and provide clear evidence that epigenetic changes like methylation are related to salivary gland carcinogenesis.     

Genetic and epigenetic alteration profiles for multiple genes in salivary gland carcinomas

As combinations of genetic and/or epigenetic alterations occurring during salivary gland carcinogenesis are largely unknown, we here analyzed 36 salivary gland carcinomas (SGCs) for changes in INK4a/ARF, RB1, p21, p27, PTEN, p53, MDM2 and O6-MGMT genes using methylation specific PCR (MSP), loss of heterozygosity (LOH) assays and mutational analysis with immunohistochemistry (IHC), as well as histone H3 and H4 acetylation status. The RB1 gene was found to be the most frequently methylated (41.7% of cases), while methylation of p27(Kip1) and O6-MGMT were less frequent 8.3% and 5.6%, respectively). Two other genes, p21(Waf1) and PTEN, were unmethylated in the SGCs examined. RB1 methylation significantly correlated with loss of expression as determined by IHC (P=0.03), and also a poor prognosis (P=0.02). p53 mutations were found in 8 cases (22.2%), coupled with p14ARF hypermethylation in two cases. LOH in INK4a/ARF and the RB1 locus was observed in 33.3% and 28.6% of the lesions, respectively. There was no correlation between 9p21 LOH and methylation of the INK4a/ARF gene. Promoter hypermethylation of RB1 coupled with LOH was evident in three samples immuno-negative for RB1. Acetylation of histone H3 and H4 was detected in 6 and 5 cases, respectively. These findings indicate that epigenetic silencing of tumour suppressor genes via promoter hypermethylation might be crucial for salivary gland carcinogenesis, particularly in the RB1 gene. Thus epigenetic events including methylation and acetylation as well as genetic alterations may have important contributions.     

Identification of novel isoforms of the EML4-ALK transforming gene in non-small cell lung cancer

The genome of a subset of non-small-cell lung cancers (NSCLC) harbors a small inversion within chromosome 2 that gives rise to a transforming fusion gene, EML4-ALK, which encodes an activated protein tyrosine kinase. Although breakpoints within EML4 have been identified in introns 13 and 20, giving rise to variants 1 and 2, respectively, of EML4-ALK, it has remained unclear whether other isoforms of the fusion gene are present in NSCLC cells. We have now screened NSCLC specimens for other in-frame fusion cDNAs that contain both EML4 and ALK sequences. Two slightly different fusion cDNAs in which exon 6 of EML4 was joined to exon 20 of ALK were each identified in two individuals of the cohort. Whereas one cDNA contained only exons 1 to 6 of EML4 (variant 3a), the other also contained an additional 33-bp sequence derived from intron 6 of EML4 (variant 3b). The protein encoded by the latter cDNA thus contained an insertion of 11 amino acids between the EML4 and ALK sequences of that encoded by the former. Both variants 3a and 3b of EML4-ALK exhibited marked transforming activity in vitro as well as oncogenic activity in vivo. A lung cancer cell line expressing endogenous variant 3 of EML4-ALK underwent cell death on exposure to a specific inhibitor of ALK catalytic activity. These data increase the frequency of EML4-ALK-positive NSCLC tumors and bolster the clinical relevance of this oncogenic kinase.     

Significant effect of a pre-exercise high-fat meal after a 3-day high-carbohydrate diet on endurance performance

We investigated the effect of macronutrient composition of pre-exercise meals on endurance performance. Subjects consumed a high-carbohydrate diet at each meal for 3 days, followed by a high-fat meal (HFM; 1007 ± 21 kcal, 30% CHO, 55% F and 15% P) or high-carbohydrate meal (HCM; 1007 ± 21 kcal, 71% CHO, 20% F and 9% P) 4 h before exercise. Furthermore, just prior to the test, subjects in the HFM group ingested either maltodextrin jelly (M) or a placebo jelly (P), while subjects in the HCM ingested a placebo jelly. Endurance performance was measured as running time until exhaustion at a speed between lactate threshold and the onset of blood lactate accumulation. All subjects participated in each trial, randomly assigned at weekly intervals. We observed that the time until exhaustion was significantly longer in the HFM + M (p < 0.05) than in HFM + P and HCM + P conditions. Furthermore, the total amount of fat oxidation during exercise was significantly higher in HFM + M and HFM + P than in HCM + P (p < 0.05). These results suggest that ingestion of a HFM prior to exercise is more favorable for endurance performance than HCM. In addition, HFM and maltodextrin ingestion following 3 days of carbohydrate loading enhances endurance running performance.