Nucleated red blood cells participate in myocardial regeneration in the toad Bufo Gargarizan Gargarizan.

Heart regeneration is negligible in humans and mammals but remarkable in some ectotherms. Humans and mammals lack nucleated red blood cells (NRBCs), while ectotherms have sufficient NRBCs. This study used Bufo gargarizan gargarizan, a Chinese toad subspecies, as a model animal to verify our hypothesis that NRBCs participate in myocardial regeneration. NRBC infiltration into myocardium was seen in the healthy toad hearts. Heart needle-injury was used as an enlarged model of physiological cardiomyocyte loss. It recovered quickly and scarlessly. NRBC infiltration increased during the recovery. Transwell assay was done to in vitro explore effects of myocardial injury on NRBCs. In the transwell system, NRBCs could infiltrate into cardiac pieces and could transdifferentiate toward cardiomyocytes. Heart apex cautery caused approximately 5% of the ventricle to be injured to varying degrees. In the mildly to moderately injured regions, NRBC infiltration increased and myocardial regeneration started soon after the inflammatory response; the severely damaged region underwent inflammation, scarring, and vascularity before NRBC infiltration and myocardial regeneration, and recovered scarlessly in four months. NRBCs were seen in the newly formed myocardium. Enzyme-linked immunosorbent assay and Western blotting showed that the levels of tumor necrosis factor-α, interleukin- 1ß, 6, and11, cardiotrophin-1, vascular endothelial growth factor, erythropoietin, matrix metalloproteinase- 2 and 9 in the serum and/or cardiac tissues fluctuated in different patterns during the cardiac injury-regeneration. Cardiotrophin-1 could induce toad NRBC transdifferentiation toward cardiomyocytes in vitro. Taken together, the results suggest that the NRBC is a cell source for cardiomyocyte renewal/regeneration in the toad; cardiomyocyte loss triggers a series of biological processes, facilitating NRBC infiltration and transition to cardiomyocytes. This finding may guide a new direction for improving human myocardial regeneration.

Identification of differentially expressed genes in salivary adenoid cystic carcinoma cells associated with metastasis.

INTRODUCTION: Salivary adenoid cystic carcinoma (SACC) is a frequent type of salivary gland cancer which is characterized by slow growth but high incidence of distant metastasis. We aimed to identify therapeutic targets which are associated with metastasis of SACC. MATERIAL AND METHODS: Total RNA was isolated from a low metastatic SACC cell line (ACC-2) and a highly metastatic SACC cell line (ACC-M), which was screened from ACC-2 by combination of in vivo selection and cloning in vitro. Then the total RNA was subjected to microarray analysis. Differentially expressed genes (DEGs) were screened from ACC-M compared with ACC-2, followed by Gene Ontology function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Function annotation for DEGs also was performed. A protein-protein interaction network (PPI) was constructed for DEGs. RESULTS: A total of 1128 DEGs were identified from ACC-M cells compared with ACC-2 cells. Both up- and down-regulated DEGs were enriched in different functions in biological process (BP), cellular component (CC) and molecular function (MF). Additionally, down-regulated DEGs were mainly enriched in "Apoptosis" and "Cytokine-cytokine receptor interaction" pathways which involved IFN-α1, NTRK1 and TGF-ß1. In the PPI network, PIK3CA, PTPN11 and PIK3R1 had a number of nodes greater than 10. CONCLUSIONS: Transforming growth factor ß1 might play a pivotal role during lung metastasis of SACC and be selected as a candidate target for treatment of metastatic SACC. IFNA1, NTRK1 and PIK3CA were also associated with tumor metastasis.

Distinct Regulatory Changes Underlying Differential Expression of TEOSINTE BRANCHED1-CYCLOIDEA-PROLIFERATING CELL FACTOR Genes Associated with Petal Variations in Zygomorphic Flowers of Petrocosmea spp. of the Family Gesneriaceae.

CYCLOIDEA (CYC)-like genes, belonging to the plant-specific TCP transcription factor family that is named after TEOSINTE BRANCHED1 (TB1) from maize (Zea mays), CYC from Antirrhinum majus, and the PROLIFERATING CELL FACTORS (PCF) from rice (Oryza sativa), have conserved dorsal identity function in patterning floral zygomorphy mainly through specific expression in dorsal petals of a flower. Their expression changes are usually related to morphological diversity of zygomorphic flowers. However, it is still a challenge to elucidate the molecular mechanism underlying their expression differentiation. It is also unknown whether CINCINNATA (CIN)-like TCP genes, locally controlling cell growth and proliferation, are involved in the evolution of floral zygomorphy. To address these questions, we selected two closely related species, i.e. Petrocosmea glabristoma and Petrocosmea sinensis, with distinct petal morphology to conduct expression, hybridization, mutant, and allele-specific expression analyses. The results show that the size change of the dorsal petals between the two species is mainly mediated by the expression differentiation of CYC1C and CYC1D, while the shape variation of all petals is related to the expression change of CIN1. In reciprocal F1 hybrids, the expression of CYC1C, CYC1D, and CIN1 conforms to an additive inheritance mode, consistent with the petal phenotypes of hybrids. Through allele-specific expression analyses, we find that the expression differentiation of these TCP genes is underlain by distinctly different types of regulatory changes. We suggest that highly redundant paralogs with identical expression patterns and interspecific expression differentiation may be controlled by remarkably different regulatory pathways because natural selection may favor different regulatory modifications rather than coding sequence changes of key developmental genes in generating morphological diversity.

Silencing of polo-like kinase 2 increases cell proliferation and decreases apoptosis in SGC-7901 gastric cancer cells.

Polo­like kinase 2 (PLK2) is a serine/threonine protein kinase, which has vital roles during mitosis and the centrosome cycle. In acute myeloblastic leukemia and hepatocarcinogenesis, PLK2 acts as a tumor suppressor; however, the function of PLK2 in gastric cancer remains to be elucidated. In the present study, PLK2 was overexpressed in gastric cancer tissues and three types of gastric cancer cells, SGC­7901, MKN­45 and BGC­823. Transfection of SGC­7901 gastric cancer cells with small interfering (si)RNA against PLK2 exerted no effect on the ratio of cells at different stages of the cell cycle compared with that of the untransfected and control siRNA­transfected cells. In addition, silencing of PLK2 significantly enhanced the growth of SGC­7901 cells through inhibiting apoptosis. Furthermore, apoptosis­associated genes Bax and caspase 3 were found to be downregulated at the protein level. In conclusion, these results suggested that PLK2 may act as a tumor suppressor in gastric cancer, therefore indicating its therapeutic potential.

Mir-126 inhibits growth of SGC-7901 cells by synergistically targeting the oncogenes PI3KR2 and Crk, and the tumor suppressor PLK2.

MicroRNA (miRNA)-126 (miR-126) was reported to be downregulated and to act as a tumor suppressor in cancers of the lung, cervix, bladder and prostate. However, the functions of miR-126 in gastric cancer appear to be diverse and are largely unknown. MiR-126 was reported to act as a tumor suppressor by targeting the Crk gene, or as an oncogene by targeting the SOX2 gene in gastric cancer. We identified that the expression of miR-126 was decreased in gastric cancer cell lines and tissues. PLK2, a tumor suppressor gene, was directly regulated by miR-126 in SGC-7901 cells. Overexpression of miR-126 not only suppressed the growth and clone formation of SGC-7901 cells, but also induced apoptosis in vitro, whereas inhibition of miR-126 slightly promoted SGC-7901 cell proliferation. The cell cycle was not affected by miR-126. Moreover, miR-126 suppressed tumor growth in vivo in a xenograft model. PLK2, PI3KR2 and Crk were regulated by miR-126 in SGC-7901 cells. We infer that the functions of miR-126 in gastric cancer depend on synergistic targeting balance between oncogenes and anti-oncogenes. Our study indicates that miR-126 is a tumor suppressor, which in the future may become a therapeutic target for gastric cancer.

Transfusion of autologous late-outgrowth endothelial cells reduces arterial neointima formation after injury.

AIMS: Late-outgrowth endothelial cells (OECs) exist in blood and other organs. We aimed to explore whether and how OECs participate in re-endothelialization and prevent vascular neointima formation after injury. METHODS AND RESULTS: Rabbit bone marrow OECs were cultured for 4 weeks to increase their numbers. Transfusion of autologous OECs (2 × 106-1 × 107/kg) soon after rabbit ear central artery injury reduced the increase in intima area and the decrease in lumen area observed at days 14 and 28. Transfusion of autologous OECs (1 × 107/kg) ameliorated some early (days 2 and 7) inflammatory and angiogenic responses (local and systemic) to the injury. Red fluorescence was seen within 7 days after transfusion of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labelled acetylated low-density lipoprotein (Dil-acLDL)-incorporated OECs, and 1 h after perfusion of the isolated rabbit ear with Ringer-Locke solution containing Dil-acLDL-incorporated OECs, in the injured rabbit ear central artery. After transfusion of 5-bromo-2'-deoxyuridine (BrdU) incorporated autologous OECs, BrdU-positive cells appeared in the injured artery intima at day 3 and were present in the rescued artery endothelium at day 28. The OECs, ranging from 5%-15% of vascular smooth muscle cells (VSMCs), and the OEC-conditioned medium (5-15%) both inhibited VSMC proliferation and migration in vitro and regulated the arrangement of VSMCs. The VSMCs were helpful for OECs to form tubes in vitro. CONCLUSION: Circulating OECs participate in re-endothelialization directly and inhibit VSMC migration and proliferation by a paracrine pathway; transfusion of large numbers of autologous OECs soon after vascular injury may prevent neointima formation.

Exogenous phosphatidylethanolamine induces apoptosis of human hepatoma HepG2 cells via the bcl-2/Bax pathway.

AIM: To investigate the signaling pathways implicated in phosphatidylethanolamine (PE)-induced apoptosis of human hepatoma HepG2 cells. METHODS: Inhibitory effects of PE on human hepatoma HepG2 cells were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle, apoptosis and mitochondrial transmembrane potential (DeltaPsi m) were analyzed by flow cytometry. Immunocytochemical assay and Western blotting were used to examine Bcl-2, Bax and caspase-3 protein levels in HepG2 cells treated with PE. RESULTS: PE inhibited the growth of HepG2 cells in a dose- and time- dependent manner. It did not affect the cell cycle, but induced apoptosis. PE significantly decreased DeltaPsi m at 0.25, 0.5 and 1 mmol/L, respectively, suggesting that PE induces cell apoptosis by decreasing the mitochondrial transmembrane potential. The Bcl-2 expression level induced by different concentrations of PE was lower than that in control groups. However, the Bax expression level induced by PE was higher than that in the control group. Meanwhile, PE increased the caspase-3 expression in a dose- and time-dependent manner. CONCLUSION: Exogenous PE induces apoptosis of human hepatoma HepG2 cells via the bcl-2/bax pathway.

[Effects of progesterone on intracellular free Ca2+ concentration in the spermatozoa of fertile men and patients with unexplained infertility].

OBJECTIVE: To investigate the difference in the responsiveness of intracellular free Ca2+ concentration ([Ca2+]i) to progesterone in the spermatozoa of normal fertile men and patients with unexplained infertility. METHODS: Nine normal fertile men and 10 patients with unexplained infertility were selected in this study. After swim-up separation of the motile fraction and 2-hour in vitro capacitation, the spermatozoa were loaded with the fluorescent calcium indicator Fluo-3/AM (8.85 micromol/L) for 40 minutes away from the light, and then the sperm suspension was mixed with equal amount of 20% gelatin to immobilize the spermatozoa. The basal intracellular free [Ca2+]i and that induced by 10 micromol/L progesterone in the individual sperm were assessed by laser scanning confocal microscopy. RESULTS: The infertile patients had a significantly lower basal level of [Ca2+]i in the capacitated sperm than the fertile men (P < 0.01). The sperm from the normal controls responded to progesterone by exhibiting a rapid but transient rise in [Ca2+]i, with the peak level significantly higher than the basal level (P < 0.05), while those from the infertile patients by showing a slight increase, with no significant difference between the peak and basal levels (P > 0.05). Both the peak of the progesterone-induced [Ca2+]i and its increase amplitude expressed as the difference between the peak and basal levels were significantly higher in the normal fertile group than in the infertile patients (P < 0.01). CONCLUSION: The responsiveness of [Ca2+]i to progesterone is reduced in the spermatozoa of patients with unexplained infertility, which suggests a functional defect in the non-genomic sperm membrane progesterone receptor responsible for calcium influx.

Gene expression profile of salivary adenoid cystic carcinoma associated with perineural invasion.

Adenoid cystic carcinoma (ACC) is a common salivary gland malignancy characterized by slow but progressive clinical course, proclivity for hematogenous spread and perineural invasion (PNI) that exhibits inherent resistance to complete surgical resection, systemic chemotherapy and conventional radiotherapy. The molecular alterations that underlie its PNI are poorly characterized. We report the combined use of laser capture microdissection (LCM) and high-throughput cDNA microarray to monitor in vivo gene expression profile of salivary ACC and to correlate the profile with PNI. Consecutive section staining with hematoxylin & eosin was applied to 15 cancerous tissues, among which 6 were judged as PNI. Pure cancer cells adjacent to the nerve tracts from 6 cancerous tissues judged as PNI were laser captured, and pure cancer cells from the same 6 tumors distant from the nerve tracts were also procured. Total RNA was extracted, amplified and subjected to cDNA microarray-based expression analysis. The patterns of gene expression were verified by quantitative real-time PCR and immunohistochemistry. As to the result of 6 arrays, a total of 53 genes were identified as being 2-fold or more differentially expressed in PNI cancer cell group as compared to non-PNI cancer cell control. Out of the 53 genes found consistently differentially expressed, 38 were up-regulated and 15 down-regulated. The combined use of LCM and cDNA microarray analysis provides a powerful new approach to monitor the in vivo molecular events of PNI in salivary ACC. These identified novel genes deserve further investigations to elucidate their clinicopathological significance.

[Construction of eukaryotic expression vector of congenital long QT syndrome related HERG gene A561V mutation and its expression in HEK293 cells].

OBJECTIVE: To investigate the protocol of the construction of HERG gene mutations, an A561V mutation which was detected in a Chinese congenital long QT syndrome (LQTS) family had been constructed and expressed in vitro. METHODS: The A561V cloning vector PGEM-HERG-A561V was constructed by quick site-directed mutagenesis PCR. The A561V expressive vector pcDNA3-HERG-A561V was constructed by restriction enzymes. pRK5-GFP was cotransfected with pcDNA3-HERG-A561V or wild type pcDNA3-HERG into HEK293 cells by Superfect transfection reagent. The protein was measured by immunofluorescence. RESULTS: Direct sequence analyses revealed a C to T transition at position 1682. The A561V mutation was correctly combined to eukaryotic expressive vector pcDNA3 and expressed in HEK293 cells. The protein of mutation was expressed in cytoplasm and cellular membrane while the wild type gene was expressed only on cellular membrane. CONCLUSION: The protocol can be used successfully to construct and express HERG A561V mutation and it forms the basement of the further study on functions of mutation.

[Inhibition of the expression of p42MAPK in HeLa cell line by RNA interference].

OBJECTIVE: To screen for siRNAs that inhibit the expression of p42(MAPK) in HeLa cell line. METHODS: Three p42(MAPK) siRNAs and one random siRNA were synthesized using Silencer siRNA Construction Kit, and labeled with Cy-3 for measurement of transfection effect. SiRNAs were transfected into HeLa cells by Lipofectamin 2000. The expression of p42(MAPK) was analyzed by Western blot. The biological effect of siRNAs on HeLa cell growth was monitored by MTT and flow cytometry. RESULTS: Two siRNAs (siRNA-2 and siRNA-3) among three tested were identified to be able to downregulate the p42(MAPK) expression. A concurrent growth retardation of HeLa cell line was observed in comparison with the control. CONCLUSION: Inhibition of p42(MAPK) expression with siRNA technique can inhibit the proliferation of HeLa cells.

[The inhibition of 20(R)-ginsenoside Rg3 on the expressions of angiogenesis factors proteins in human lung adenocarcinoma cell line A549 and HUVEC304 cell].

OBJECTIVE: To study the effect of 20( R)-ginsenoside Rg3 on the expressions of angiogenesis factors proteins (VEGF,bFGF, MMP-2) in human lung adenocarcinoma cell line A549 and HUVEC304 cell. METHOD: The cell lines of A549 and HUVEC304 were cultured with 20(R)- Rg3. The gray scale and positive rate of VEGF, bFGF, MMP-2 were detected by immunohistochemistry. The differential expressions of genes were studied by DNA microarray. RESULT: The positive rate of VEGF protein in A549 cell decreased significantly as compared with the control group ( P = 0.03). The gray scales of VEGF, Flt, KDT proteins in both A549 cell lines and HUVEC 304 cell lines decreased ( P = 0.05). Gray scale of MMP-2 also decreased in A549 cell lines. The result of differential expressions of genes of A549 cell lines showed that 14 genes were down-regulated and 10 genes were up-regulated. CONCLUSION: The Chinese materia medica of 20( R)-Rg3 can inhibit the expression of angiogenesis factors proteins via several target genes in both tumour cell and vascular endothelial cell.

[Effects of NGF and estrogens on human hair follicle in vitro].

OBJECTIVE: To observe the effects of NGF, estrogens and minoxidil on the growth of human hair follicle in vitro. METHODS: In a model of human hair follicle in vitro, the follicle was separately treated with the NGF, estrogens and minoxidil. The growth of the hair follicle was measured in length with an eyepiece micrometer. The effects of the NGF, estrogens and minoxidil were evaluated by measuring the rates of incorporation of 3H-TdR of DNA synthesis. RESULTS: The growth of the human hair follicle was showing significantly faster in the 100 ng/ml NGF and 125 micrograms/ml minoxidil groups, compared with the control (P < 0.05), but the growth was significantly inhibited in the 0.5 microgram/ml 17 beta-E2 group (P < 0.05). There was no difference shown for the growth of the hair follicle in the group mixed with 100 ng/ml NGF and 0.5 microgram/ml 17 beta-E2 (P > 0.05). The rates of incorporation of 3H-TdR in the groups were shown that the results just correlated with the results of the above-mentioned method. CONCLUSIONS: The 100 ng/ml NGF and 125 micrograms/ml minoxidil could increase the growth of human hair follicle while the 0.5 microgram/ml 17 beta-E2 could inhibit it. The 100 ng/ml NGF could neutralized the effect of the 0.5 microgram/ml 17 beta-E2.