MicroRNA-19a-3p suppresses invasion and metastasis of prostate cancer via inhibiting SOX4.

OBJECTIVE: To explore the role of microRNA-19a-3p in regulating invasion, metastasis and EMT (epithelial mesenchymal transition) of prostate cancer (PCa) cells, as well as its underlying mechanism. MATERIALS AND METHODS: MicroRNA-19a-3p mimic and negative control plasmid were first constructed. After transfection of microRNA-19a-3p mimic or negative control in DU145 cells, expression levels of microRNA-19a-3p and SOX4 were detected by quantitative Real-time-polymerase chain reaction (qRT-PCR) and Western blot. The regulatory effects of microRNA-19a-3p on migration and invasion of DU145 cells were detected by wound healing assay and transwell assay, respectively. Protein levels of matrix metalloproteinase-2 (MMP2), matrix metalloproteinase-9 (MMP9), N-cadherin, Vimentin, alpha-smooth muscle actin (α-SMA) and E-cadherin in DU145 cells transfected with microRNA-19a-3p mimic or negative control were detected by Western blot. RESULTS: Overexpression of microRNA-19a-3p inhibited protein level of SOX4 in DU145 cells. The migration and invasion of DU145 cells were inhibited after transfection of microRNA-19a-3p mimic. Protein levels of MMP2, MMP9, N-cadherin, Vimentin and α-SMA were downregulated, whereas E-cadherin was upregulated after microRNA-19a-3p overexpression. CONCLUSIONS: MicroRNA-19a-3p inhibits migration, invasion and EMT of PCa cells via inhibiting SOX4.

[Effects of different sulfonylureas on the warm-up phenomenon in diabetes patients with coronary artery disease].

Objective: To explore the different effects of chronic treatment with glibenclamide and gliclazide on the warm-up phenomenon in diabetes patients with coronary artery disease. Methods: A total of seventy-one patients with chronic stable angina and diabetes who were positive for exercise test and was proven that the stenosis degree was 70%-90% in at least one major branch through coronary angiogram were included into the study.They were divided into three groups, diet control group (DMD), glibenclamide group (DMG1) and gliclazide group (DMG2), according to the treatment of diabetes.All of the patients underwent two bicycle exercise tests (EX) at 15-minute interval.Parameters including ischaemic threshold (the rate-pressure product at 1-mm ST-segment depression, RPP), time to ischaemic (the time to 1 mm ST-segment depression, T-STD), the maximum ST-segment depression (STDmax) and exercise duration (ED) were recorded respectively. Results: In group DMD, T-STD and ED were prolonged [(360±83) s vs (409±80) s, P<0.001] and [(518±90) s vs (549±96) s, P=0.001], STDmax were shortened [(1.91±0.43) mm vs (1.60±0.36) mm, P<0.001], and RPP was increased [(180±27) beats·min(-1)·mmHg·10(2) vs (195±28) beats·min(-1)·mmHg·10(2), P<0.001] as the parameters during EX2 were compared with those during EX1. In group DMG1, there was no statistic difference in these indexes except that ED was prolonged [(458±70) s vs (472±66) s, P=0.045] when those of EX2 and EX1were compared. In the group DMG2, all the analyzed variables improved significantly during two sequential exercise tests as the results in the group DMD except that ischaemic threshold was not increased [(199±41) beats·min(-1)·mmHg·10(2) vs (211±39) beats·min(-1)·mmHg·10(2), P=0.071]. Conclutions: Warm-up phenomenon is abolished in diabetic patients with stable angina treated with glibenclamide, partially preserved in gliclazide-treated patients. and the KATP channel may be involved in those different effects. Gliclazide should be the safer choice for the patients with diabetes and chronic stable angina.

Correlation between chromosome 1p/19q status and VEGF mRNA expression in gliomas.

The chromosome 1p/19q deletion has been reported as a good prognosis marker for gliomas. However, the detection of 1p/19q status alone in glioma patients is not sufficient. The identification of a combination of molecular factors could effectively enhance the prediction accuracy. Thus far, the potential correlation between the 1p/19q status and vascular endothelial growth factor (VEGF) expression has not been elucidated. The level of VEGF mRNA expression in the tumor and the adjacent normal tissues was determined by real-time polymerase chain reaction. The 1p/19q status of glioma patients was determined by fluorescence in situ hybridization. The association between the 1p/19q status and VEGF mRNA expression, as well as the glioma grade, was evaluated. A higher VEGF mRNA expression level was observed in gliomas, compared to matched normal tissues (P < 0.01). The 1p/19q status was significantly correlated with glioma grade (P = 0.018) and VEGF mRNA expression in the tissues (P = 0.005). A higher percentage of patients with high-grade gliomas displayed an intact 1p/19q and higher VEGF mRNA expression than those with low-grade gliomas. A survival analysis revealed that patients (with high- and low-grade gliomas) with intact 1p/19q and higher VEGF mRNA expression showed a shorter overall survival time. Moreover, tissue VEGF mRNA expression and WHO grade were found to be independent risk factors for gliomas. In conclusion, the 1p/19q status and VEGF mRNA expression in tissues could be used in combination to predict the prognosis of gliomas.

First Report of Botrytis cinerea Causing Gray Mold of Tobacco in Guizhou Province of China.

Tobacco (Nicotiana tabacum L.) is a leafy, annual, solanaceous plant grown commercially for its leaves. Guizhou Province produces more than 30% of the total Chinese tobacco crop. In July 2010, a disease was observed in a commercial field of 5-month-old N. tabacum plants in Bijie, Guizhou in southwestern China. Symptoms first appeared on the leaves as small spots that later increased in size and developed into expanded, dark brown lesions covered with green-gray spore masses. Lesions expanded rapidly under cool, humid conditions. Isolates of Botrytis cinerea were collected from diseased leaves with typical symptoms. Diseased leaf samples were washed with distilled water three times, placed in a moist chamber, and incubated at 25°C in darkness for 48 h to encourage sporulation. Spores produced on leaves were transferred to individual agar discs (5 mm in diameter) with an inoculating needle and then the agar discs were transferred to potato dextrose agar (PDA) and incubated at 25°C for 7 days. Fungal colonies were at first colorless and later became gray to brown when the conidia differentiated. The size of conidia ranged from 5.0 to 9.5 × 6.5 to 12.5 µm (average 7.3 × 8.7 µm) based on 50 spore measurements. Microsclerotia produced in the culture were round or irregular and ranged from 1.2 to 3.0 × 1.0 to 2.5 mm (average 2.1 × 2.0 mm). The pathogen was identified as B. cinerea Pers.:Fr on the basis of morphology and sequence of ITS1-5.8s-ITS2 region of rDNA amplified by PCR using universal primers ITS-1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS-4 (5'-TCCTCCGCTTATTGATATGC-3'). The sequence (GenBank Accession No. HQ902163) exactly matched the sequences of two Botryotinia fuckeliana (anamorph B. cinerea) accessions, (e.g., GenBank Accession Nos. HM849615.1 and HM849047.1). Koch's postulates were conducted by wound inoculating five tobacco leaves (cv. K326) after surface disinfesting them with 5% NaOCl. Plugs of the fungus (5 mm in diameter) obtained from the colony margins were transferred onto 3 × 3 mm wounds made with a needle on the surface of five sterilized leaves. Inoculated leaves were incubated at 25°C, 100 to 120 µE·m-2·s-1, relative humidity >80%, and 16 h light per day for disease development. Typical symptoms developed on leaves within 7 days after inoculation. The pathogen was reisolated from affected leaves but not from the noninoculated control leaves. Botrytis gray mold blight has been recorded on N. tabacum in New Zealand, the United Kingdom, and northern China (1-3). However, to our knowledge, this is the first report of Botrytis blight on N. tabacum in Guizhou Province of China and the disease must be considered in existing disease management practices. References: (1) W. Brian et al. Mol. Plant Pathol. 8:561, 2007. (2) A. G. Mcleod et al. N. Z. J. Crop Hortic. Sci/Exp. Agric. 12:866, 1958. (3) Z. Y. Zhang. Page 37 in: Flora Fungorum Sinicorum. Vol. 26. Science Press, Beijing, 2006.

Long-term survival of cardiac allografts induced by cyclophosphamide combined with CTLA4Ig-gene transfer mediated by adenoviral vector.

There is a need to achieve donor-specific tolerance in clinical organ transplantation, where potential benefits remain overshadowed by chronic rejection and the side-effects of long-term immunosuppressive therapy. It is known that the mature immune system in mice can be reprogrammed to accept a foreign graft as if it was "self". The AdCTLA4Ig-mediated gene transfer (SC) + cyclophosphamide (CP) treatment alone prolongs allograft survival but does not induce tolerance. However, in our study, the AdCTLA4Ig-mediated gene transfer combined with SC + CP treatment yielded significantly prolonged mean survival times (149.7 +/- 18.0 days), while those in the untreated or AdLacZ treated mice were rejected in normal fashion (5.3 +/- 0.5 and 5.2 +/- 0.4 days, respectively), and survival in the AdCTLA4Ig or SC + CP treated groups were 45.7 +/- 9.6 or 50.2 +/- 5.3 days, respectively. In conclusion, a protocol of AdCTLA4Ig + SC + CP improved the survival of DA-->LEW cardiac allografts.

CTLA4-Fas ligand gene transfer mediated by adenovirus induce long-time survival of murine cardiac allografts.

BACKGROUND: Fas ligand gene transfer to induce peripheral allograft tolerance in animal models has shown controversial results. The immunosuppression effects mediated by engineered FasL depend on whether alloreactive T cells are selectively deleted. In the present study, we tested the feasibility of a strategy to induce long-time survival by fusing CTLA4-FasL gene transfer in vivo. METHODS: Cardiac allografts from DA(RT-1(a)) rats were transplanted heterotopically into the abdomens of LEW(RT-1(1)) rats. Plaque units (5x10(9)) of either AdCTLA4-FasL, AdCTLA4Ig, or AdEGFP were administered via the portal vein immediately after cardiac transplantation. The frequencies of helper T lymphocyte precursors (HTLp) and cytotoxic T lymphocyte precursors (CTLp) were determined by a combined single limiting dilution assay on days 5 and 20 after transplantation. RESULTS: Cardiac allograft survival was significantly prolonged by either AdCTLA4-FasL or AdCTLA4Ig treatment(mean survival times [MST] of 71.0 +/- 3.7 and 45.7 +/- 2.4, respectively, n = 6) compared with untreated hosts or animals treated with AdEGFP(MST of 5.7 +/- 0.5 and 5.2 +/- 0.4, respectively, n = 6). In addition, treatment with AdCTLA4-FasL led to significantly prolonged allograft survival compared with AdCTLA4Ig treatment. Furthermore, the frequencies of HTLp and CTLp on day 20 among rats treated with AdCTLA4-FasL was lower than those on day 5, whereas frequencies of HTLp and CTLp on day 20 were similar with those on day 5 in the other groups. CONCLUSION: These results suggest that administration of an adenovirus encoding fusion CTLA4-FasL gene to rat recipients effectively decreased the size of alloreactive T cells and induced long-term survival of cardiac allografts.

Antileukemic effect of interleukin-7-transduced bone marrow stromal cells in mice following allogeneic T-cell-depleted bone marrow transplantation.

Impaired immune reconstitution following allogeneic bone marrow transplantation (BMT) remains a major obstacle to its clinical application. In this study, interleukin (IL)-7-transduced bone marrow stromal cells (MSC-IL7, 1 x 10(6)/mouse) were transfused into lethally irradiated C57BL/6 recipient mice. By day 40 after transplantation, the recipient mice were challenged with the lymphoma cell line EL4. MSC-IL7 co-transplantation protected recipient mice from leukemic mortality (MST >120 days after BMT vs mean survival time (MST) 70 days in the PBS group) It enhance the PFC count and DTH responses of recipients after transplantation. In conclusion, MSC mediated IL-7 gene therapy and may be a more feasible strategy to restore immune function following allo-TCD-BMT.

Keratin 17 mutation in pachyonychia congenita type 2 with early onset sebaceous cysts.

BACKGROUND: Pachyonychia congenita (PC) is a group of autosomal dominant ectodermal dysplasias caused by mutations in four differentiation-specific keratin genes. Two major clinical subtypes of PC have been generally recognized. Symmetrically thickened fingernails and toenails are the defining characteristic of PC type 2 (PC-2) with onset at infancy. Pilosebaceous cysts are the best hallmark of PC-2, but they usually occur at puberty. OBJECTIVES: To report a Chinese pedigree of PC-2 with unusually early onset sebaceous cysts and to explore the genetic mutation and its phenotype. METHODS: Exon 1 of keratin 17 was amplified by polymerase chain reaction (PCR) from genomic DNA from the three patients in the pedigree, the proband, his half-sister and his younger son, two unaffected members in the pedigree and 50 unrelated and unaffected people. PCR products were directly sequenced to detect the mutation. RESULTS: Direct sequencing of the PCR products revealed a heterozygous 275A-->G mutation in all three affected members. This mutation predicts the substitution of asparagine by serine in codon 92 (N92S) located in the 1A domain of keratin 17. CONCLUSIONS: Mutation in the 1A domain of keratin 17 underlies the affected members' phenotype, PC-2 with early onset sebaceous cysts and late-onset thickened fingernails and toenails. The onset of the cysts is very early in some people within this family and the age at onset of thickened fingernails and toenails is variable within the family, implying the existence of modifying factors.

Effect of Salvia miltiorrhiza Bunge injection on anticardiolipin antibody production induced by beta2 glycoprotein.

AIM: To explore the therapeutic effect and the mechanism of Chinese herbs on antiphospholipid syndrome (APS) by observing the effect of Salvia miltiorrhiza Bunge injectio (SmBI) on anticardiolipin antibody (aCL) induced by beta2 glycoprotein I (beta2-GP I). METHODS: Sixty female mice randomly fell into 6 groups: group A, B, C, D was injected through abdominal cavity with different dosage of SmBI daily; after 14 d, group A, B, C, E was immunized with 150 microg of purified human beta2-GP I in complete Freund's adjuvant subcutaneously; group F as control. The titre of aCL were detected by enzyme linked immunosorbent assay; subsets of T cell were grouped by streptavidin-biotin complex technique; and the activity of IL-2 was measured by MTT chromatometry. RESULTS: (1) Compared with group E, the absorbance (A) of aCL in group A, B, and C was decreased (P < 0.05 or P < 0.01). By linear correlation, the dosage is negatively correlated with the A values of aCL in 1, 2, and 3 weeks (P < 0.01). (2) Compared with group E, TH/TS ratio was reduced in group A, B, and C (P < 0.05 or P < 0.01); there is no significant differences between group D and F (P>0.05). By linear correlation, the dosage is negatively correlated with TH/TS ratio (P < 0.01). (3) Compared with E, the activity of IL-2 in group B and C decreased significantly (P < 0.01). By linear correlation, there is negative correlation between dosage and IL-2 activity (P < 0.01). There is no significant difference between D and F (P > 0.05). (4) There is positive correlation between TH/TS ratio and IL-2 activity in different dilutions (P<0.01). CONCLUSION: The mechanism of suppressive effect of SmBI on aCL induced by beta2-GP I may be realized by resuming the elevated TH/TS ratio and IL-2 activity. The state that SmBI have no effect on normal mice indicates that SmBI has selective immunoregulative functive.

Modulation of the binding affinity of myelopoietins for the interleukin-3 receptor by the granulocyte colony-stimulating factor receptor agonist.

Myelopoietins (MPOs) are a family of recombinant chimeric proteins that are both interleukin-3 (IL-3) receptor and granulocyte colony-stimulating factor (G-CSF) receptor agonists. In this study, MPO molecules containing one of three different IL-3 receptor agonists linked with a common G-CSF receptor agonist have been examined for their IL-3 receptor binding characteristics. Binding to the alpha-subunit of the IL-3 receptor revealed that the affinity of the MPO molecules was 1.7-3.4-fold less potent than those of their individual cognate IL-3 receptor agonists. The affinity decrease was reflected in the MPO chimeras having approximately 2-fold slower dissociation rates and 2.7-5.5-fold slower association rates than the corresponding specific IL-3 receptor agonists alone. The affinity of binding of the MPO molecules to the heteromultimeric alphabeta IL-3 receptor expressed on TF-1 cells was either 3-, 10-, or 42-fold less potent than that of the individual cognate IL-3 receptor agonist. Biophysical data from nuclear magnetic resonance, near-UV circular dichroism, dynamic light scattering, analytical ultracentrifugation, and size exclusion chromatography experiments determined that there were significant tertiary structural differences between the MPO molecules. These structural differences suggested that the IL-3 and G-CSF receptor agonist domains within the MPO chimera may perturb one another to varying degrees. Thus, the differential modulation of affinity observed in IL-3 receptor binding may be a direct result of the magnitude of these interdomain interactions.