Upregulation of CBP by PLY can cause permeability of blood-brain barrier to increase meningitis.

BACKGROUND: Streptococcus pneumoniae causes many human diseases including bacterial meningitis. Previous study proposed that pneumolysin (PLY), a cytotoxin from pneumococcus, is related to the infection across blood-brain barrier (BBB). However, the mechanism of how PLY break through BBB remains elusive. The present study showed that PLY can increase the permeability of BBB both in vitro and in vivo in our experiments. RESULTS: Further we found out that PLY leads to the high expression of CERB-binding protein (CBP) which can lead to releasing of tumor necrosis factor α then enhance apoptosis of cells which is a significant factor leading to permeabilization of BBB. CONCLUSION: Our findings demonstrate that CBP plays an important role in the pneumococcus infection in the brain and could be a potential therapeutic target against pneumococcal meningitis.

Novel CDH1 germline mutations identified in Chinese gastric cancer patients.

AIM: To give a comprehensive report of E-cadherin gene (CDH1) variations in a population at a high risk for gastric cancer (GC). METHODS: The samples consisted of 178 men and 58 women with a mean age of 62.3 ± 9.4 years and an age range of 30-84 years. A total of 240 cancer-free controls were recruited (mean age of 61.8 ± 10.1 years, age range of 26-82 years). Samples were screened for CDH1 germline mutations by high-resolution melting analysis or directly sequencing. Luciferase reporter assay, RNA splicing assay and bioinformatic analysis were used to evaluate the effect of mutations. RESULTS: Four novel CDH1 sequence alterations were identified in GC patients including a G>T transition 49 bp before the start codon; a three-nucleotide deletion, c.44_46del TGC; one missense mutation, c.604G>A (V202I); and one variation in the intron, c.1320+7A>G. In addition, polymorphism frequencies were observed for CDH1-164delT, -161C>A, -73A>C, c.48+6C>T, c.48+62_48+63delinsCGTGCCCCAGCCC, c.894C>T (A298A), c.1224G>A (A408A), c.1888C>G (L630V), c.2076T>C (A692A), and c.2253C>T (N751N) which is similar to the data reported in http://www.ncbi.nlm.nih.gov/projects/SNP/. RNA splicing analysis suggested that the c.1320+7A>G and c.1224G>A variations did not affect exon splicing ability. Luciferase reporter assay demonstrated that the c.-49T variation might be helpful for E-cadherin transcription, though the increase in transcription activity is limited (only 33%). SIFT score and PolyPhen analysis both demonstrated that the L630V missense mutation probably damages protein function, while the V202I variant does not. CONCLUSION: This study reveals novel mutations in sporadic GC patients which had been poorly investigated for susceptibility genes.

[Influence of microsatellite instability on survival of patients with sporadic colorectal cancer in China].

OBJECTIVE: To investigate the role of microsatellite instability(MSI) in Chinese sporadic coloretal cancer. METHODS: A total of 146 patients with colorectal cancer were treated surgically from August 2004 to September 2006 in the Third Affiliated Hospital of Nanjing University of Traditional Chinese Medicine. Data were collected prospectively. Univariate and multivariable analyses were performed for parameters such as age, gender, tumor location, differentiation, MSI, tumor type, lymph node metastasis, TNM stage, and survival. RESULTS: Follow-up was available in 134 patients including telephone call and office visit. MSI(P=0.029), tumor type(P=0.000), TNM stage(P=0.000) were independently associated with survival on Cox regression model. There were 26 patients with MSI, and the 1-, 3-, and 5-year survival rates were 100%, 92.3%, and 92.3%, respectively. The remaining 108 patients had microsatellite stable tumor, and the 1-, 3-, and 5-year survival rates were 96.3%, 72.2%, and 63.5%, respectively. The difference was statistically significant(P=0.016). CONCLUSION: Microsatellite instability is an important factor associated with patient survival in Chinese sporadic colorectal cancer.

[Tea polyphenol inhibits colorectal cancer with microsatellite instability by regulating the expressions of HES1, JAG1, MT2A and MAFA].

OBJECTIVE: To investigate the mechanism of tea polyphenol in inhibiting microsatellite instability (MSI) of colorectal cancer. METHODS: Using LoVo cells and SW480 cells treated with aqueous solution of tea polyphenol, cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) method, changes in microsatellite sequences were detected by genescan method and changes in gene expression of LoVo cells were detected by illumina expression arrays and quantitative real-time polymerase chain reaction (PCR). RESULTS: The proliferation inhibition rates of LoVo and SW480 cells treated with tea polyphenol increased with the increasing of drug concentration and showed an increasing tendency with time. The proliferation inhibition rate of LoVo cells with tea polyphenol was higher than that of SW480 cells, and there was a significant difference in the proliferation inhibition rates at 24 h, 72 h and one week. The microsatellite sequence of LoVo cells treated with tea polyphenol remained stable. The gene expression arrays and quantitative real-time PCR suggested that tea polyphenol inhibited the gene expressions of MT2A, MAFA, HES1 and JAG1 nearly two-fold over controls. It was also found that tea polyphenol inhibited the BAX and p38 genes with a more than two-fold difference but did not significantly inhibit the nuclear factor-κB pathway. CONCLUSION: Tea polyphenol significantly inhibited the proliferation of MSI colorectal cancer cells and stably maintained the microsatellite state in MSI colorectal cancer. Tea polyphenol inhibited the gene expressions of HES1, JAG1, MT2A and MAFA, up-regulated the gene expression of BAX and down-regulated that of P38. Further research is required to investigate how these pathways are interrelated.

[Establishment of colostomy orthotopic transplantation mice and study on their biological characteristics].

OBJECTIVE: To establish a colorectal cancer colostomy orthotopic transplantation mice model. METHODS: A colostomy was preformed in BALB/C nu-nu nude mice. After two weeks, when the stoma healed, tumor tissues developed from Lovo cells were implanted into the submucosa of the stoma. When tumor grew up to 5 mm, fluorouracil(5-FU, 20 mg/kg) was administrated by intraperitoneal injection. Tumor developed at the colostomy was observed and its biological characteristics and behaviour were evaluated. RESULTS: Colostomy was performed in 10 mice and stoma healed at two weeks. Ten colostomies developed detectable tumor in two to three weeks. Three to five weeks later, the tumors grew up to 5 mm. Survival time of mice injected with 5-FU was(15.2+/-3.7) weeks (ranged:11-21 weeks), and the survival time of the no-treatment group was(12.3+/-2.8) weeks(ranged:9-19 weeks). The difference was statistically significant(P=0.001). The rate of mesenteric metastasis was 1/5 and 2/5 in the treatment and no-treatment group respectively. CONCLUSION: Colostomy orthotopic transplantation mice model is an ideal mice model with the advantages of having high success rate, visualization of implanted tumor in living animal, long survival time and significant tumor response to common chemotherapeutic agent.

[Anticancer effects of tea polyphenols on colorectal cancer with microsatellite instability in nude mice].

OBJECTIVE: To study the anticancer effects of tea polyphenols on colorectal cancer with microsatellite instability (MSI) in nude mice and to explore its mechanism. METHODS: A colostomy was performed on the caecum of nude mice. Tumor fragments collected from the subcutaneous tumor of hMSH2-absence colon carcinoma Lovo cell line were surgically implanted onto the submucosa of the caecum during colostomy to establish the model. Then, the nude mice were divided into untreated group and 50, 75 and 100 mg/kg tea polyphenols groups. The mice in tea polyphenols-treated groups were given intra-abdominal injection of 50, 75 and 100 mg/kg tea polyphenols respectively. The inhibition rates of tumors were calculated, and microsatellite instability (MSI) and the alteration of transforming growth factor-beta1 (TGF-beta1), TGF-beta2 and insulin-like growth factor (IGF) were detected by Genescan method at different times after the injection. RESULTS: The tumor volumes of the three groups began to decrease at the 1st week and decreased most greatly from 2 to 3 weeks after treatment, and then the tumors tended to increase. The study found that tea polyphenols could inhibit the tumor growth. The tumor inhibition rates in the three treated groups were significantly higher than those in untreated group 1, 2, 3 and 4 weeks after treatment (P<0.01). Detection of MSI showed that the colorectal tumor in the untreated group presented with four MSI signs, including BAT-25, D2S123, D5S346 and D17S250, and TGF-beta1, TGF-beta2, IGF expressions. After using the tea polyphenols, the microsatellite tended to become stable. CONCLUSION: Tea polyphenols can inhibit the mismatch-repair-gene deficient colorectal cancer in nude mice by down-regulating the microsatellite instability.

[Effect of DNA-PKcs antisense oligodeoxynucleotides on radiosensitivity of nasopharyngeal carcinoma cell lines with different p53 function statuses].

BACKGROUND & OBJECTIVE: DNA-dependent protein kinase (DNA-PK) can repair DNA double-strand break. This study was to observe the effect of DNA-PKcs antisense oligodeoxynucleotides (ASODN) on the radiosensitivity of nasopharyngeal carcinoma (NPC) cell lines with normal or abnormal p53 functions. METHODS: DNA-PKcs ASODN was transfected into CNE-1 and CNE-1-wtp53 cells. These cells were irradiated with 0, 0.5, 1, 2, 4, 6, or 8 Gy X-ray. Cell survival was determined by clonogenic assay. The parameters D0, Dq, and N for the single-hit multitarget model and the parameters alpha, beta, alpha/beta, and SF2 for the linear-quadratic model were calculated to evaluate the changes of radiosensitivity. RESULTS: The alpha values before DNA-PKcs ASODN transfection were 0.03 in CNE-1 cells and 0.05 in CNE-1-wtp53 cells; the alpha values after transfection were 0.04 in CNE-1 cells and 0.27 in CNE-1-wtp53 cells. The SF2 before transfection were 0.73 in CNE-1 cells and 0.50 in CNE-1-wtp53 cells; the SF2 after transfection were 0.45 in CNE-1 cells and 0.21 in CNE-1-wtp53 cells. The D0 before transfection were 2.08 Gy in CNE-1 cells and 1.13 Gy in CNE-1-wtp53 cells; the D0 after transfection were 1.07 Gy in CNE-1 cells and 0.83 Gy in CNE-1-wtp53 cells. The Dq before transfection were 2.04 Gy in CNE-1 cells and 1.36 Gy in CNE-1-wtp53 cells; the Dq after transfection were 1.24 Gy in CNE-1 cells and 0.73 Gy in CNE-1-wtp53 cells. The parameter alpha of CNE-1 cells was increased after DNA-PKcs ASODN transfection, but the parameters SF2, D0, and Dq were decreased after transfection. CONCLUSION: DNA-PKcs ASODN could enhance the radiosensitivity of CNE-1 cells regardless of p53 function status.

[Screening the hereditary nonpolyposis colorectal cancer by revised Bethesda guideline: a cohort study of 110 cases].

OBJECTIVE: To study the value of screening hereditary nonpolyposis colorectal cancer (HNPCC) by the revised Bethesda guideline and the rate of HNPCC in colorectal cancer (CRC). METHOD: Tumor tissues and normal colorectal mucous membrane tissues were collected from 110 successive cases with CRC, 66 males and 42 females, aged 60.8 (26 - 94). Fluorescence multiplex polymerase chain reaction was used to detect the microsatellite instability (MSI). The peripheral blood samples were collected from the patients with MSI, genomic DNA was extracted, and PCR and DNA sequencing were used to detect the germline mutations of hMSH2, hMSH6, and hMLH1. RESULTS: Twenty-three out of the 110 patients (20.9%), 12 males and 22 females, aged 57 (47 - 94), had MSI. Seven germline mutations were found in these 23 MSI patients, accounting for 6.4% among the 110 CRC patients, including 3 cases of hMSH2 mutation, 3 cases of hMSH6 mutation, and 1 case of mutation of hMLH1. CONCLUSION: Screened by revised Bethesda guideline, the rate of MSI CRC is 20.9% and the rate of HNPCC is 6.4%. The missence germline mutations of hMSH2 and hMSH6 are more common in the Chinese patients with CRC.

Radiotherapy-related typing in 842 patients in canton with nasopharyngeal carcinoma.

PURPOSE: The aim of this study was to propose the clinical radiotherapy-related typing and to summarize proportional distribution of radiotherapy-related types of nasopharyngeal carcinoma (NPC). METHODS AND MATERIALS: A total of 842 cases of NPC were randomly selected. According to 5-year follow-up results after radiotherapy, NPC was subdivided into four types: Type I (no primary and regional recurrence and no distant metastasis), Type II (primary or regional recurrence and no distant metastasis), Type III (no primary and regional recurrence, and distant metastasis), and Type IV (primary or regional recurrence, and distant metastasis). Proportion of the four types and relationship between this typing and Zhi-guang Xie typing were analyzed. RESULTS: Distribution of radiotherapy-related types of NPC were 50.6%, 23.2%, 20.7%, and 5.6% for Types I, II, III, and IV, respectively. For Types D and AD of Zhi-guang Xie typing system and Stage III and IV of the 1992 Fuzhou staging system, the proportion of Type III was greater than that of Type II; and for Type A and Stage I and II, there was a larger proportion of Type II than that of Type III. CONCLUSION: Radiotherapy-related typing, as a new clinical subclassification, could be supplementary for previous clinical typing and staging.

Preliminary results of a randomized study (NPC-9902 Trial) on therapeutic gain by concurrent chemotherapy and/or accelerated fractionation for locally advanced nasopharyngeal carcinoma.

PURPOSE: To compare the benefit achieved by concurrent chemoradiotherapy (CRT) and/or accelerated fractionation (AF) vs. radiotherapy (RT) alone with conventional fractionation (CF) for patients with T3-4N0-1M0 nasopharyngeal carcinoma (NPC). METHODS AND MATERIALS: All patients were irradiated with the same RT technique to > or =66 Gy at 2 Gy per fraction, conventional five fractions/week in the CF and CF+C (chemotherapy) arms, and accelerated six fractions/week in the AF and AF+C arms. The CF+C and AF+C patients were given the Intergroup 0099 regimen (concurrent cisplatin plus adjuvant cisplatin and 5-fluorouracil). RESULTS: Between 1999 and April 2004, 189 patients were randomly assigned; the trial was terminated early because of slow accrual. The median follow-up was 2.9 years. When compared with the CF arm, significant improvement in failure-free survival (FFS) was achieved by the AF+C arm (94% vs. 70% at 3 years, p = 0.008), but both the AF arm and the CF+C arm were insignificant (p > or = 0.38). Multivariate analyses showed that CRT was a significant factor: hazard ratio (HR) = 0.52 (0.28-0.97), AF per se was insignificant: HR = 0.68 (0.37-1.25); the interaction of CRT by AF was strongly significant (p = 0.006). Both CRT arms had significant increase in acute toxicities (p < 0.005), and the AF+C arm also incurred borderline increase in late toxicities (34% vs. 14% at 3 years, p = 0.05). CONCLUSIONS: Preliminary results suggest that concurrent chemoradiotherapy with accelerated fractionation could significantly improve tumor control when compared with conventional RT alone; further confirmation of therapeutic ratio is warranted.

The effect of p21 antisense oligodeoxynucleotides on the radiosensitivity of nasopharyngeal carcinoma cells with normal p53 function.

OBJECTIVE: p21(WAF1/CIP1) is transcriptionally activated by p53 and is required for G1 to S phase progression. p21 plays a critical role in DNA repair after DNA damage. Thus, cells with defective p21 may result in an enhancement of radiation induced apoptosis and improved radiosensitivity. We tested the hypothesis that p21 antisense oligodeoxynucleotides (p21 AS ODNs) can be used to reduce p21 expression level and increase radiosensitivity in CNE-1-wtp53 nasopharyngeal carcinoma cell line with normal p53 function. METHODS AND MATERIALS: The p21 antisense oligodeoxynucleotides (p21 AS ODNs) and the random control oligodeoxynucleotides (p21 RD ODNs) were synthesized. p21 AS ODNs sequence: 5'-TGTCATGCTGGTCTGCCGCC-3'; p21 RD ODNs sequence: 5'-CCGGTGAACGAGCGAGCACA-3'. p21 AS ODNs and p21 RD ODNs were transfected into CNE-1-wtp53 nasopharyngeal carcinoma cell line. The protein expression levels of P21 were evaluated using Western blotting analysis. Cell cycle progression and apoptotic cells were assessed by flow cytometric analysis. The clonogenic survival assay was performed to determine the survival fraction. The parameters D0, Dq, and N for the single-hit multitarget model and the parameters alpha, beta, alpha/beta, and SF2 for the linear-quadratic model were calculated. BALB/c nude mice were used to investigate the effect of p21 AS ODNs on the radiosensitivity of nasopharyngeal xenografts in vivo. RESULTS: p21 AS ODNs were detected mainly in plasma with fluorescence microscopy investigation. P21 protein level dramatically decreased and the amount of apoptotic cells increased in p21 AS ODNs transfected cells than in p21 RD ODNs transfected cells after irradiation. The percentage of G1 arrest decreased in p21 AS ODNs transfected cells 24 h after radiation, then G2 arrest decreased 48 h after radiation. The values of D0, Dq, SF2 decreased and alpha value increased in p21 AS ODNs transfected cells than in control cells. The inhibition rate in tumor xenografts exposed to X ray of 10 Gy alone was 39.1%, while it was 51.4% in xenografts injected with p21 AS ODNs before exposure to radiation. Unfortunately, there was no significant difference between these two groups (P < 0.05). CONCLUSION: p21 Antisense oligodeoxynucleotides led to inhibition of P21 protein expression, loss of G1 arrest, increase of apoptosis in CNE-1-wtp53 nasopharyngeal carcinoma cell line in vitro and inhibited tumor growth in vivo. Antisense oligodeoxynucleotides may become a promising strategy to enhance radiosensitivity in nasopharyngeal carcinoma cells with normal p53 function.

[Clinical study on radiotherapy-related typing of nasopharyngeal carcinoma].

OBJECTIVE: To advance a new system of clinico-radiotherapeutic typing of nasopharyngeal carcinoma (NPC) and compare it with the typing system of Xie Zhiguang. METHODS: 842 patients with NPC, 559 males and 283 females, aged 48 (15-76), that had undergone CT and were treated with radical radiotherapy alone as the first therapeutic measure at least 5 years ago, were followed up for 5 years. Their cancers were divided into 4 types according to whether recurrence occurred in the primary focus and/or regional lymph nodes and whether distant metastasis occurred. The cancers without recurrence in the primary foci and regional lymph nodes and without distant metastasis was defined as radiosensitive and not liable to metastasize type (type I), those with recurrence in the primary foci and/or regional lymph noses but without distant metastasis were defined as radio-resistant and not liable to metastasize type (type II), those without recurrence in the primary foci and/or regional lymph nodes but with distant metastasis were defined as radiosensitive and liable to metastasize type (type III), and those with recurrence in the primary foci and/or regional lymph nodes and distant metastasis as well were defined as radio-resistant and liable to metastasize type (type IV). The distribution of these four clinico-radiotherapeutic types and the relation between them and the Xie Zhi-guang types: ascending type (type A), descending type (type D), and ascending and descending type (type AD). RESULTS: (1) The percentages of the Types I, II, III, and IV were 50.6% (426 cases), 23.2% (195 cases), 20.7% (174 cases), and 5.6% (47 cases) respectively. (2) 264 of the 842 patients could be typed according to Xie Zhi-guang typing criteria: 65 were defined as type A (24.6%), 61 (23.1%) as type D and 138 (52.3%) as type AD. Among the 65 patients of type A 58.5% were of type I, 29.2% were of type II, 10.8% were of type III, and 1.5% were of type IV; among the type D patients 47.5% were of type I, II 9.8% were of type, 36.1% were of type III, and 6.6% were of type IV; and among the type AD patients 34.8% were of type I, 26.8% were of type II, 33.3% were of type III, and 5.1% were of type IV. (3) 307 patients were at the early stage of Fuzhou clinical classification 1992 (stages I and II) among which 191 were of type I (62.2%), and 65 were of type II (21.2%), 38 were of the type III (12.4%), and 13 were of the type IV (4.2%). Among the 535 patients at the late stage (stages III and IV), 235 were of the type I (43.9%), 130 of the type II (24.3%), 136 were of the type III (25.4%), and 34 were of the type IV (6.4%). CONCLUSION: There are four radiotherapy-related types in NPC with the constituent ratio as: radiosensitive and not liable to metastasize type (type I) > radio-resistant and not liable to metastasize type (type II) > radiosensitive and liable to metastasize type (type III) > radio-resistant and liable to metastasize type (type IV). Among the NPC patients of the type D and type AD according to the Xie Zhiguang classification and the NPC patients at the advanced stage (stages III and IV) of the Fuzhou staging system 1992, the proportion of radiosensitive and liable to metastasize type (type III) exceeds that of the radio-resistant and not liable to metastasize type (type II). The radiotherapy-related typing system is a supplement to the clinical typing and staging of NPC.

[Retrospective analysis of 934 nasopharyngeal carcinoma patients treated with conventional external beam radiotherapy alone].

OBJECTIVE: To analyze the clinical outcome of 934 primary nasopharyngeal carcinoma treated with conventional external beam radiotherapy alone. METHODS: 34 patients were treated from Jan. 1, 1999 to Dec. 31, 1999. The radiation fields were delineated according to the CT/MRI imaging findings on disease extent. Two lateral opposing isocentric portals with customized blockings were used for the nasopharynx and upper neck. The dose delivered to tumor in the nasopharynx was 68-70 Gy/2 Gy fraction/7 weeks. The doses delivered to the neck was 60-70 Gy/6-7 weeks for patients with positive lymph nodes and 50 Gy/5 weeks for the patients with negative lymph node. RESULTS: The 1-, 2-, 3- and 4-year overall survival rate (OS) was 89.5%, 81.9%, 78.1% and 75.7%, and metastasis-free survival rate (MFS) was 84.0%, 77.2%, 74.4% and 72.0%, respectively. The 1-, 2-, 3- and 4-year disease-free survival rate (DFS) was 80.8%, 73.1%, 68.5% and 65.1%, and the relapse-free survival rate (RFS) was 95.5%, 92.7%, 90.3% and 87.3%, respectively. The overall failure rate was 30.9% (289/934). At the end of the radiotherapeutic course, the percentage of residual disease was 14.6%. The 4-year loco-regional recurrence and distant metastasis rates after radiotherapy were 7.2% and 9.2% with a median time of 19.3 months and 12.8 months. CONCLUSION: It may be helpful to improve radiotherapy curative effect when the target is individually designed through improving irradiation technique according to CT/MRI findings and by shortening the overall course time, enhancing irradiation dose and strictly implementing QA/QC measures.

Gender differences in ANG II levels and action on multiple K+ current modulation pathways in diabetic rats.

Gender differences were studied in ventricular myocytes from insulin-deficient (Type 1) diabetic rats. Cells were obtained by enzymatic dispersion of hearts from control male and female rats and from rats made diabetic with streptozotocin (100 mg/kg) 7-14 days before experiments. ANG II content, measured by ELISA, was augmented in diabetic males but unaltered in diabetic females. In diabetic ovariectomized females, ANG II levels were augmented as in males. ANG II affects multiple cellular pathways including activation of protein kinase C (PKC) and several tyrosine kinases as well as inhibition of protein kinase A (PKA). The involvement of these pathways in modulating outward K(+) currents was studied. Transient and sustained outward K(+) currents were measured using the whole cell voltage-clamp method. In males, these currents are attenuated under diabetic conditions but are augmented by the ANG II-converting enzyme inhibitor quinapril. Activation of PKA by 8-bromo-cAMP enhanced both K(+) currents in cells from diabetic males. The augmentation of these currents by quinapril was blocked when PKA inhibition was maintained with the Rp isomer of 3',5'-cyclic monophosphorothioate. Inhibition of tyrosine kinases by genistein also augmented K(+) currents in cells from diabetic males. Action potentials were abbreviated by 8-bromo-cAMP and genistein. However, both genistein and 8-bromo-cAMP had no effect on K(+) currents in cells from diabetic females. In cells from ovariectomized diabetic females, 8-bromo-cAMP and genistein enhanced these K(+) currents as in males. Inhibition of PKC augmented the transient and sustained K(+) currents in cells from diabetic males and females. A contribution of non-ANG II-dependent activation of PKC is suggested. These results describe some of the mechanisms that may underlie gender-specific differences in the development of cardiac disease and arrhythmias.

Sex differences in the modulation of K+ currents in diabetic rat cardiac myocytes.

A transient (Ipeak) and a sustained (Isus) outward K+ current were measured, using whole-cell voltage-clamp methods, in isolated rat ventricular myocytes obtained by enzymatic dispersion. A comparison was made between male and female rats following induction of (insulin-deficient) diabetes with streptozotocin (STZ). In control (non-diabetic) rats, both currents were smaller in cells obtained from females, as compared to males (P<0.005). However, whereas inducing diabetes in male rats significantly attenuated both Ipeak and Isus (P<0.005), Ipeak was unchanged in female diabetic rats. Isus was significantly (P<0.005) reduced, but the extent of reduction was smaller (P<0.02) than in males. The formation of angiotensin II (ATII) or endothelin-1 (ET-1) was blocked using inhibitors of angiotensin-converting enzyme (ACE) and endothelin-converting enzyme (ECE), respectively. In cells from diabetic males both inhibitors significantly (P<0.005) enhanced K+ currents. In contrast, no effect was observed in cells from female diabetic rats. However, in ovariectomized (Ovx) diabetic females the in vitro inhibition of ATII and ET-1 formation augmented the two K+ currents, but not when oestradiol was administered in vivo prior to cell isolation. In cells from diabetic males, incubation with 100 nM 17beta-oestradiol significantly (P<0.005) enhanced both Ipeak and Isus. This effect was blocked if ATII or ET-1 was added to the medium. These results show that autocrine modulation of K+ currents by renin-angiotensin and endothelin systems is attenuated or absent in female diabetic rats. Oestradiol plays a key role in reducing this modulation. These results may underlie some of the sex differences associated with development of cardiac arrhythmias.