E(y)1/TAF9 mediates the transcriptional output of Notch signaling in Drosophila.

Transcriptional activation of Notch signaling targets requires the formation of a ternary complex that involves the intracellular domain of the Notch receptor (NICD), DNA-binding protein Suppressor of Hairless [Su(H), RPBJ in mammals] and coactivator Mastermind (Mam). Here, we report that E(y)1/TAF9, a component of the transcription factor TFIID complex, interacts specifically with the NICD-Su(H)-Mam complex to facilitate the transcriptional output of Notch signaling. We identified E(y)1/TAF9 in a large-scale in vivo RNA interference (RNAi) screen for genes that are involved in a Notch-dependent mitotic-to-endocycle transition in Drosophila follicle cells. Knockdown of e(y)1/TAF9 displayed Notch-mutant-like phenotypes and defects in target gene and activity reporter expression in both the follicle cells and wing imaginal discs. Epistatic analyses in these two tissues indicated that E(y)1/TAF9 functions downstream of Notch cleavage. Biochemical studies in S2 cells demonstrated that E(y)1/TAF9 physically interacts with the transcriptional effectors of Notch signaling Su(H) and NICD. Taken together, our data suggest that the association of the NICD-Su(H)-Mastermind complex with E(y)1/TAF9 in response to Notch activation recruits the transcription initiation complex to induce Notch target genes, coupling Notch signaling with the transcription machinery.

CAF-1 promotes Notch signaling through epigenetic control of target gene expression during Drosophila development.

The histone chaperone CAF-1 is known for its role in DNA replication-coupled histone deposition. However, loss of function causes lethality only in higher multicellular organisms such as mice and flies, but not in unicellular organisms such as yeasts, suggesting that CAF-1 has other important functions than histone deposition during animal development. Emerging evidence indicates that CAF-1 also has a role in higher order chromatin organization and heterochromatin-mediated gene expression; it remains unclear whether CAF-1 has a role in specific signaling cascades to promote gene expression during development. Here, we report that knockdown of one of the subunits of Drosophila CAF-1, dCAF-1-p105 (Caf1-105), results in phenotypes that resemble those of, and are augmented synergistically by, mutations of Notch positive regulatory pathway components. Depletion of dCAF-1-p105 leads to abrogation of cut expression and to downregulation of other Notch target genes in wing imaginal discs. dCAF-1-p105 is associated with Suppressor of Hairless [Su(H)] and regulates its binding to the enhancer region of E(spl)mß. The association of dCAF-1-p105 with Su(H) on chromatin establishes an active local chromatin status for transcription by maintaining a high level of histone H4 acetylation. In response to induced Notch activation, dCAF-1 associates with the Notch intracellular domain to activate the expression of Notch target genes in cultured S2 cells, manifesting the role of dCAF-1 in Notch signaling. Together, our results reveal a novel epigenetic function of dCAF-1 in promoting Notch pathway activity that regulates normal Drosophila development.

Histone chaperone CAF-1: essential roles in multi-cellular organism development.

More and more studies have shown chromatin remodelers and histone modifiers play essential roles in regulating developmental patterns by organizing specific chromosomal architecture to establish programmed transcriptional profiles, with implications that histone chaperones execute a coordinating role in these processes. Chromatin assembly factor-1 (CAF-1), an evolutionarily conserved three-subunit protein complex, was identified as a histone chaperone coupled with DNA replication and repair in cultured mammalian cells and yeasts. Interestingly, recent findings indicate CAF-1 may have important regulatory roles during development by interacting with specific transcription factors and epigenetic regulators. In this review, we focus on the essential roles of CAF-1 in regulating heterochromatin organization, asymmetric cell division, and specific signal transduction through epigenetic modulations of the chromatin. In the end, we aim at providing a current image of facets of CAF-1 as a histone chaperone to orchestrate cell proliferation and differentiation during multi-cellular organism development.

Drosophila CAF-1 regulates HP1-mediated epigenetic silencing and pericentric heterochromatin stability.

Chromatin assembly factor 1 (CAF-1) was initially characterized as a histone deliver in the process of DNA-replication-coupled chromatin assembly in eukaryotic cells. Here, we report that CAF-1 p180, the largest subunit of Drosophila CAF-1, participates in the process of heterochromatin formation and functions to maintain pericentric heterochromatin stability. We provide evidence that Drosophila CAF-1 p180 plays a role in both classes of position effect variegation (PEV) and in the expression of heterochromatic genes. A decrease in the expression of Drosophila CAF-1 p180 leads to a decrease in both H3K9 methylation at pericentric heterochromatin regions and the recruitment of heterochromatin protein 1 (HP1) to the chromocenter of the polytene chromosomes. The artificial targeting of HP1 to a euchromatin location leads to the enrichment of Drosophila CAF-1 p180 at this ectopic heterochromatin, suggesting the mutual recruitment of HP1 and CAF-1 p180. We also show that the spreading of heterochromatin is compromised in flies that have reduced CAF-1 p180. Furthermore, reduced CAF-1 p180 causes a defect in the dynamics of heterochromatic markers in early Drosophila embryos. Together, these findings suggest that Drosophila CAF-1 p180 is an essential factor in the epigenetic control of heterochromatin formation and/or maintenance.

High percentages of peripheral blood T-cell activation in childhood Hodgkin's lymphoma are associated with inferior outcome.

The aims of this study were to investigate the activation of T lymphocytes in peripheral blood from children with Hodgkin's lymphoma (HL) and explore their roles for prognosis in HL. A cohort of 52 newly diagnosed children with HL during the past 10 years was enrolled for analysis in this study. Peripheral blood samples of the patients were acquired before treatment in our hospital, and T-cell subsets were detected by a four-color flow cytometer. CD4+ T cells and CD4+/CD8+ T-cell ratio decreased significantly in patients with HL vs. healthy controls. CD8+ T cells, CD3+CD4+HLA-DR+ T cells, and CD3+CD8+HLA-DR+ T cells increased markedly in patients with HL vs. healthy controls. Receiver-operating characteristic (ROC) curve analysis showed that CD3+CD4+HLA-DR+ T cells and CD3+CD8+HLA-DR+ T cells each distinguished the high-risk group from the low- and intermediate-risk group. The area under the ROC curve for predicting high-risk patients was 0.795 for CD3+CD4+HLA-DR+ T cell and 0.784 for CD3+CD8+HLA-DR+ T cell. A comparison of peripheral blood T-cell subsets that responded differently to therapy showed significantly higher percentages of CD3+CD4+HLA-DR+ T cells and CD3+CD8+HLA-DR+ T cells in patients who achieved complete remission compared to those who did not achieve complete remission. In addition, high percentages of both CD3+CD4+HLA-DR+ T cells and CD3+CD8+HLA-DR+ T cells were associated with inferior event-free survival. Peripheral immune status may be related to disease severity in HL. CD3+CD4+HLA-DR+ T cells and CD3+CD8+HLA-DR+ T cells may be a novel indicator for risk stratification of HL and may be an independent risk factor for inferior outcome in childhood HL.

Regulation of gap junctional communication by astrocytic mitochondrial K(ATP) channels following neurotoxin administration in in vitro and in vivo models.

It is known that neuronal ATP-sensitive potassium (K(ATP)) channels and astrocytic gap junctions (GJs) are involved in the mechanism underlying neurodisorders. The K(ATP) channels exist also in glial cells, and the objective of this study was to determine whether the astrocytic K(ATP) channels exert their effect on neurotoxin-induced neurodysfunction through regulating the astrocytic GJ function. The results showed that diazoxide, a selective mitochondrial K(ATP) (mitoK(ATP)) channel opener, enhanced the GJ coupling, but 5-hydroxydecanoate, a selective mitoK(ATP) channel blocker that significantly inhibits GJ coupling in vitro did not. Activation of astrocytic mitoK(ATP) channels alleviated kainic acid-induced dysfunction of GJ intercellular communication. Finally, activation of mitoK(ATP) channels improved the astrocytic GJ coupling in the hippocampus after seizures due to the colabeling of GJ subunit connexin 43 and connexin 45 with glial marker and was increased substantially by the administration of diazoxide. Western blot demonstrated that the mitoK(ATP) channels regulated the expression of connexin 43 (P2; active form) and connexin 45 in the epileptic hippocampus. These findings demonstrate that activation of astrocytic mitoK(ATP) channels improves the GJ function in astrocytes, indicating that the effect of the astrocytic mitoK(ATP) channels on neurotoxin-induced neurodysfunction might be, in part, through the regulation of the GJ-coupled spatial buffering in the hippocampus.

Deletion analysis of SMN1 and NAIP genes in Southern Chinese children with spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a disorder characterized by degeneration of lower motor neurons and occasionally bulbar motor neurons leading to progressive limb and trunk paralysis as well as muscular atrophy. Three types of SMA are recognized depending on the age of onset, the maximum muscular activity achieved, and survivorship: SMA1, SMA2, and SMA3. The survival of motor neuron (SMN) gene has been identified as an SMA determining gene, whereas the neuronal apoptosis inhibitory protein (NAIP) gene is considered to be a modifying factor of the severity of SMA. The main objective of this study was to analyze the deletion of SMN1 and NAIP genes in southern Chinese children with SMA. Here, polymerase chain reaction (PCR) combined with restriction fragment length polymorphism (RFLP) was performed to detect the deletion of both exon 7 and exon 8 of SMN1 and exon 5 of NAIP in 62 southern Chinese children with strongly suspected clinical symptoms of SMA. All the 32 SMA1 patients and 76% (13/17) of SMA2 patients showed homozygous deletions for exon 7 and exon 8, and all the 13 SMA3 patients showed single deletion of SMN1 exon 7 along with 24% (4/17) of SMA2 patients. Eleven out of 32 (34%) SMA1 patients showed NAIP deletion, and none of SMA2 and SMA3 patients was found to have NAIP deletion. The findings of homozygous deletions of exon 7 and/or exon 8 of SMN1 gene confirmed the diagnosis of SMA, and suggested that the deletion of SMN1 exon 7 is a major cause of SMA in southern Chinese children, and that the NAIP gene may be a modifying factor for disease severity of SMA1. The molecular diagnosis system based on PCR-RFLP analysis can conveniently be applied in the clinical testing, genetic counseling, prenatal diagnosis and preimplantation genetic diagnosis of SMA.

Author Correction: Transient Scute activation via a self-stimulatory loop directs enteroendocrine cell pair specification from self-renewing intestinal stem cells.

In the version of this Article originally published, the author had misnumbered the reference citations in the Methods, using numbers 1-14 instead of 46-59. These errors have now been corrected in all online versions of the Article.

Transient Scute activation via a self-stimulatory loop directs enteroendocrine cell pair specification from self-renewing intestinal stem cells.

The process through which multiple types of cell-lineage-restricted progenitor cells are specified from multipotent stem cells is unclear. Here we show that, in intestinal stem cell lineages in adult Drosophila, in which the Delta-Notch-signalling-guided progenitor cell differentiation into enterocytes is the default mode, the specification of enteroendocrine cells (EEs) is initiated by transient Scute activation in a process driven by transcriptional self-stimulation combined with a negative feedback regulation between Scute and Notch targets. Scute activation induces asymmetric intestinal stem cell divisions that generate EE progenitor cells. The mitosis-inducing and fate-inducing activities of Scute guide each EE progenitor cell to divide exactly once prior to its terminal differentiation, yielding a pair of EEs. The transient expression of a fate inducer therefore specifies both type and numbers of committed progenitor cells originating from stem cells, which could represent a general mechanism used for diversifying committed progenitor cells from multipotent stem cells.

[Association between clinical manifestations of infants with human cytomegalovirus infection and glycoprotein B genotype].

OBJECTIVE: To investigate the association between the clinical manifestations of infants with human cytomegalovirus (HCMV) infection and glycoprotein B (gB) genotype. METHODS: Urine samples were obtained from 107 symptomatic infants with HCMV infection confirmed by fluorescence quantitative PCR, 70 male and 37 female, aged 5 d-8 months, and 25 asymptomatic infants with HCMV infection, 16 male and 9 female, aged 21 d-7 months. A fragment of glycoprotein B gene was amplified by nested PCR (nPCR). HCMV gB genotyping was carried out by restriction fragment length polymorphism (RFLP), and some of the amplified DNA fragments were verified by DNA sequencing. RESULTS: Of the HCMV specimens from 107 infants, 53 were typed as gB group I, 20 as gB II, 18 as gB III, 7 as gB I and gB II, 5 as gB I and gB III, and 4 as gB II and gB III, and gB IV was not found. The HCMV gB genotype from 53 infants with hepatic function damage showed that 36 were classified as gB I, 5 as gB II, 7 as gB III, 3 as gB I and gB II, and 2 as gB I and gB III. The gB I genotype was more common among the infants with hepatic function damage (P < 0.05). The distribution of gB genotype in the asymptomatic infants was as follow: gB I, 10/25; gB II, 6/25; gB III, 8/25; and gB I and gB II, 1/25. The homology of PCR products of HCMV gB in 24 strains amplified compared with the sequences of prototype strains in GenBank was from 97.06% to 99.64%. CONCLUSION: RFLP analysis of HCMV gB genotype is definite and reliable. The gB I genotype is more common among the infants with hepatic function damage.

The Mitochondrial DNA Polymerase Promotes Elimination of Paternal Mitochondrial Genomes.

Mitochondrial DNA (mtDNA) is typically inherited from only one parent [1-3]. In animals, this is usually the mother. Maternal inheritance is often presented as the passive outcome of the difference in cytoplasmic content of egg and sperm; however, active programs enforce uniparental inheritance at two levels, eliminating paternal mitochondrial genomes or destroying mitochondria delivered to the zygote by the sperm [4-13]. Both levels operate in Drosophila [8, 12, 13]. As sperm formation begins, hundreds of doomed mitochondrial genomes are visualized within the two huge mitochondria of each spermatid. These genomes abruptly disappear during spermatogenesis. Genome elimination, which is not in the interests of the restricted genomes, is directed by nuclear genes. Mutation of EndoG, which encodes a mitochondria-targeted endonuclease, retarded elimination [8]. Here, we show that knockdown of the nuclear-encoded mtDNA polymerase (Pol γ-α), Tamas, produces a more complete block of mtDNA elimination. Tamas is found in large particles that localize to mtDNA during genome elimination. We discount a simple possible mechanism by showing that the 3'-exonuclease function of the polymerase is not needed. While DNA elimination is a surprising function for DNA polymerase, it could provide a robust nexus for nuclear control of mitochondrial genome copy number, since use of common interactions for elimination and replication might limit options for the mitochondrial genome to escape restriction. We suggest that the DNA polymerase may play this role more widely and that inappropriate activation of its elimination ability might underlie association of DNA loss syndromes with mutations of the human mtDNA polymerase [14-16].

Epigenetics and Cellular Metabolism.

Living eukaryotic systems evolve delicate cellular mechanisms for responding to various environmental signals. Among them, epigenetic machinery (DNA methylation, histone modifications, microRNAs, etc.) is the hub in transducing external stimuli into transcriptional response. Emerging evidence reveals the concept that epigenetic signatures are essential for the proper maintenance of cellular metabolism. On the other hand, the metabolite, a main environmental input, can also influence the processing of epigenetic memory. Here, we summarize the recent research progress in the epigenetic regulation of cellular metabolism and discuss how the dysfunction of epigenetic machineries influences the development of metabolic disorders such as diabetes and obesity; then, we focus on discussing the notion that manipulating metabolites, the fuel of cell metabolism, can function as a strategy for interfering epigenetic machinery and its related disease progression as well.

Activation of ATP-sensitive potassium channels prevents the cleavage of cytosolic mu-calpain and abrogates the elevation of nuclear c-Fos and c-Jun expressions after hypoxic-ischemia in neonatal rat brain.

The purpose of this study was to determine whether activation of ATP-sensitive K+ (KATP) channels with diazoxide (DIZ) is able to prevent the cleavage of cytosolic mu-calpain and abrogate the elevation of nuclear c-Fos and c-Jun protein (c-Fos, c-Jun) expressions after hypoxic-ischemia (HI) in brain. The model of hypoxic-ischemic brain injury (HIBI) was made in the 7-day-old Sprague-Dawley (SD) rats by left carotid arterial ligation and hypoxia (8% oxygen). DIZ was injected into the left lateral ventricle (5 microl, 1 mg/ml) before or post-hypoxic-ischemia (HI) insults. Western blot and computer image processing were used to detect the integrated density of nuclear c-Fos and c-Jun at 4 h and cleavage of cytosolic mu-calpain at 24 h after HI insults from cerebral cortical and hippocampal samples. Compared with HI controls (c-Fos=30.37+/-7.39 from cortical samples, 58.61+/-3.64 from hippocampal samples; c-Jun=52.48+/-14.23 from cortical samples, 35.55+/-4.73 from hippocampal samples), there was a significant down-regulation of c-Fos and c-Jun expressions from cortical and hippocampal samples in rats treated with DIZ before (c-Fos=11.10+/-4.64 from cortical samples, 4.82+/-3.38 from hippocampal samples; c-Jun=19.01+/-5.29 from cortical samples, 35.55+/-4.73 from hippocampal samples) or post- (c-Fos=18.81+/-7.93 from cortical samples, 11.33+/-7.05 from hippocampal samples; c-Jun=24.64+/-10.01 from cortical samples, 19.75+/-3.47 from hippocampal samples) HI insults. Furthermore, the ratio of 76 kD/80 kD of mu-calpain was down-regulated from cortical and hippocampal samples in rats treated with DIZ before or post-HI insults, demonstrating a significant difference compared with that observed in HI controls. Finally, the increase in DNA fragments caused by the HI injury was decreased or eliminated by the treatment with DIZ. These data suggests that activation of KATP channels by DIZ reduces the degree of mu-calpain proteolysis, and c-Fos and c-Jun expressions in immature brain may contribute to the neuroprotection of K(ATP) channel openers against HIBI.

Effects of IGF-II on promoting proliferation and regulating nitric oxide synthase gene expression in mouse osteoblast-like cell.

OBJECTIVE: To investigate the effects of insulin-like growth factor II (IGF-II) on promoting cell proliferation, regulating levels of cellular nitric oxide (NO) and mRNA transcriptions of inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) in mouse osteoblast-like cells. METHODS: Mouse osteoblastic cell line MC3T3-E1 was selected as the effective cell of IGF-II. After the cells were treated with IGF-II at different concentrations for different time duration, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay was used to examine cell proliferation, and nitrate reductase method was applied to detect NO concentrations in cell culture supernatants and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was employed to determine transcription levels of cellular iNOS and eNOS mRNAs. RESULTS: After the MC3T3-E1 cells were treated with IGF-II at concentration of 1 ng/ml for 72 h, 10 and 100 ng/ml for 24, 48 and 72 h respectively, all the MTT values increased (P<0.05 or P<0.01) with obvious dosage-time dependent pattern. NO levels of the MC3T3-E1 cells treated with 100 ng/ml IGF-II for 48 h, and with 1, 10 and 100 ng/ml IGF-II for 72 h were remarkably lower than that of the normal control, respectively (P<0.05 or P<0.01). After the cells were treated with 100 ng/ml IGF-II for 48 h cellular iNOS mRNA levels were significantly decreased (P<0.01). But the levels of eNOS mRNA in the cells treated with each of the used IGF-II dosages for different time duration did not show any differences compared with the normal control (P>0.05). CONCLUSION: IGF-II at different concentrations could promote proliferation of mouse MC3T3-E1 cell. This cell proliferation promotion was associated with the low NO levels maintained by IGF-II. Higher concentration of IGF-II could down-regulate iNOS gene expression at the level of transcription but not affect transcription of eNOS mRNA, which might be one of the mechanisms for IGF-II maintenance of the low NO levels in MC3T3-E1 cells.

[Determination of six major human herpes viruses in cerebrospinal fluid and blood of children with consensus primers].

OBJECTIVE: To identify 6 major human herpesviruses with consensus primers and to explore its clinical application. METHODS: Based on the highly-homogeneous regions of DNA polymerase gene in human herpesviruses,Two pairs of primer were synthesized. One pair was designed to amplify herpes simplex virus type 1, type 2, Epstein-Barr virus and cytomegalovirus; and another was used to amplify varicella-zoster virus or human herpesvirus 6. Virus species identification was performed by restriction enzyme digestion with BamH I and BstU I. Thirty-eight CSF specimens of clinically diagnosed viral encephalitis,and 49 blood specimens from 27 confirmed cases and 22 clinically diagnosed ones were tested for herpes virus DNA using the PCR-RFLP assay with these primers. RESULTS: Thirteen out of 38 CSF specimens (34.2%) were herpes virus positive. All blood specimens from 27 confirmed cases showed positive results, while for 22 clinically diagnosed cases 16 (72.7%) were positive. The types of herpes virus were determined using restriction enzyme digestion with BamH I and BstU I. Two CSF specimens from the patients, who were treated with aciclovir for 2 - 3 days, were still positive for herpes virus DNA by this method. None of the control blood or CSF controls were positive for herpesvirus by PCR. CONCLUSION: The PCR-RFLP method used in this study is a specific, sensitive and practicable one for diagnosis of herpes virus infection.

[Anti-inflammation and anti-oxidation effects of recombinant human superoxide dismutase on acute lung injury induced by meconium aspiration in infant rats].

OBJECTIVE: To investigate the anti-inflammation and anti-oxidation effects of recombinant human CuZn superoxide dismutase(rhSOD) on acute lung injury (ALI) induced by meconium aspiration in rats. METHODS: 1 ml/kg of 20% human newborn meconium suspension was intratracheally (IT) administrated to induce the model of ALI in 32 male Sprage-Dawley rats, and the animals were then randomized to 4 groups: 3 treatment groups with IT administration of 5, 10 and 20 mg/kg rhSOD dissolved in 1 ml/kg saline and the control group with IT administration of 1 ml/kg saline. The animals were killed after 24 h of treatments. The measurements included lung tissue wet/dry ratio, broncho-alveolar lavage fluid (BALF) protein, BALF protein/plasma protein (pulmonary permeability index, PPI),lung myeloperoxidase (MPO) and superoxide dismutase (SOD) activity, nitric oxide (NO) and 8-isoprostane levels. Lung injury score was also evaluated. RESULTS: Compared with the control group, pulmonary MPO activity, NO and 8-isoprostane levels were significantly decreased and SOD activity was markedly increased in all rhSOD treatment groups (P<0.05 or 0.01). Compared with the rhSOD 5 mg/kg group, pulmonary 8-isoprostane level was further low in the rhSOD 20 mg/kg group(P=0.01). Lung injury score was decreased in rhSOD 20 mg/kg group (P<0.05). But there were no statistically differences in lung wet/dry, BALF protein and PPI among all groups. CONCLUSION: The results suggest that a single IT dose of 5,10 or 20 mg/kg rhSOD can prevent lung damages in rats with ALI following meconium aspiration.

Effect of diazoxide on regulation of vesicular and plasma membrane GABA transporter genes and proteins in hippocampus of rats subjected to picrotoxin-induced kindling.

Epileptiform discharges and behavioral seizures may be the consequences of excess excitation from inadequate inhibitory effects associated with gamma-aminobutyric acid (GABA). GABA is taken up and accumulated in synaptic vesicles by the action of vesicular GABA transporter (VGAT) before its release into the synaptic cleft, and removed from synaptic regions by the action of transporter proteins GABA transporter-1 (GAT-1) and GABA transporter-3 (GAT-3). In this experiment, the effects of diazoxide (DIZ) on the VGAT, GAT-1 and GAT-3 mRNA and protein levels in hippocampus, and on the seizure activities of picrotoxin (PTX)-induced kindling rats were observed. DIZ caused increase in the quantity of VGAT mRNAs and proteins, and down regulation of GABA transporters GAT-1 and GAT-3 mRNAs and proteins after the PTX re-kindling. Furthermore, DIZ produced not only a prompt but also a later suppression of PTX-induced seizures. Although DIZ has effects on ATP-sensitive potassium (K(ATP)) channels when measured in vitro, our study suggests that additional mechanisms of action may involve the regulation of GABA transporters, which may aid in understanding epileptogenesis and inform investigators about future design and development of K(ATP) channel openers to treat epilepsy.

[Influence of different DNA extractions on the identification of streptococcus sanguis group by arbitrary primed polymerase chain reaction]

OBJECTIVE: To assess the influence of different DNA extractions on the identification of streptococcus sanguis group (SSG) species by arbitrary primed polymerase chain reaction (AP-PCR). METHODS: AP-PCR was used to distinguish SSG species by designing 25bp arbitrary primer 5'AAG AGA GGA GCT AGC TCT TCT TGG A 3', and the genomic DNA was extracted by 3 methods. RESULTS: There were great differences in the main band of DNA polymorphism among SSG species. The similar band could be got from the different DNA extractions in the same species. CONCLUSION: Different DNA extractions have no influence on the identification of SSG.

[Effect of adriamycin on sarcoplasmic reticulum Ca2+ -ATPase activity in cardiomyocyte of rabbits]

OBJECTIVE: To evaluate influences of regular-dose of adriamycin (ADR) on heart function and sarcoplasmic reticulum (SR) Ca2+ -ATPase in cardiomyocyte of rabbits. METHODS: Nine rabbits received intraveneous injection of ADR (2mg/kg) once a week for 8 weeks, the rabbits injected with saline were used as control group. Cardiac output (CO), blood pressure (BP), mean artery pressure (MAP), left ventricular systolic pressure( LVSP), left ventricular end-diastolic pressure (LVEDP), calcium in cardiomyocyte (MyoCa2+) of rabbits and SR Ca2+ -ATPase activity were examinated 3 weeks after the final injection. RESULTS: CO, LVSP and SR Ca2+ -ATPase activity were significantly decreased in ADR treated group compared with the control group. Conversely, LVEDP and MyoCa2+ were significantly increased in ADR treated rabbits. CONCLUSION: Heart function can be decreased by regular-dose of ADR in injection. Calcium overload in cardiomyocyte and decrease of SR Ca2+ -ATPase activity is important physiopathologic mechanism in ADR-induced impairment of heart.

Various applications of TALEN- and CRISPR/Cas9-mediated homologous recombination to modify the Drosophila genome.

Modifying the genomes of many organisms is becoming as easy as manipulating DNA in test tubes, which is made possible by two recently developed techniques based on either the customizable DNA binding protein, TALEN, or the CRISPR/Cas9 system. Here, we describe a series of efficient applications derived from these two technologies, in combination with various homologous donor DNA plasmids, to manipulate the Drosophila genome: (1) to precisely generate genomic deletions; (2) to make genomic replacement of a DNA fragment at single nucleotide resolution; and (3) to generate precise insertions to tag target proteins for tracing their endogenous expressions. For more convenient genomic manipulations, we established an easy-to-screen platform by knocking in a white marker through homologous recombination. Further, we provided a strategy to remove the unwanted duplications generated during the "ends-in" recombination process. Our results also indicate that TALEN and CRISPR/Cas9 had comparable efficiency in mediating genomic modifications through HDR (homology-directed repair); either TALEN or the CRISPR/Cas9 system could efficiently mediate in vivo replacement of DNA fragments of up to 5 kb in Drosophila, providing an ideal genetic tool for functional annotations of the Drosophila genome.