Defective expression of the μ3 subunit of the AP-3 adaptor complex in the Drosophila pigmentation mutant carmine

The adaptor protein (AP) complexes AP-1, AP-2, and AP-3 mediate coated vesicle formation and sorting of integral membrane proteins in the endocytic and late exocytic pathways in mammalian cells. A search of the Drosophila melanogaster expressed sequence tag (EST) database identified orthologs of family members mammalian medium (μ) chain families μ1, μ2, and μ3, of the corresponding AP complexes, and δ-COP, the analogous component of the coatomer (COPI) complex. The Drosophila orthologs exhibit a high degree of sequence identity to mammalian medium chain and δ-COP proteins. Northern analysis demonstrated that medium chain and δ-COP mRNAs are expressed uniformly throughout fly development. Medium chain and δ-COP genes were cytologically mapped and the μ3 gene was found to localize to a region containing the pigmentation locus carmine (cm). Analysis of genomic DNA of the cm ¹ mutant allele indicated the presence of a large insertion in the coding region of the μ3 gene and Northern analysis revealed no detectable μ3 mRNA. Light microscopy of the cm ¹ mutant showed a reduction in primary, secondary, and tertiary pigment granules in the adult eye. These findings provide evidence of a role for μ3 in the sorting processes required for pigment granule biogenesis in Drosophila.     

Cell type–specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function

Optogenetic methods have emerged as powerful tools for dissecting neural circuit connectivity, function and dysfunction. We used a bacterial artificial chromosome (BAC) transgenic strategy to express the H134R variant of channelrhodopsin-2, ChR2(H134R), under the control of cell type–specific promoter elements. We performed an extensive functional characterization of the newly established VGAT-ChR2(H134R)-EYFP, ChAT-ChR2(H134R)-EYFP, Tph2-ChR2(H134R)-EYFP and Pvalb(H134R)-ChR2-EYFP BAC transgenic mouse lines and demonstrate the utility of these lines for precisely controlling action-potential firing of GABAergic, cholinergic, serotonergic and parvalbumin-expressing neuron subsets using blue light. This resource of cell type–specific ChR2(H134R) mouse lines will facilitate the precise mapping of neuronal connectivity and the dissection of the neural basis of behavior.     

Mineralocorticoids modulate the expression of the β-3 subunit of the Na+, K+-ATPase in the renal collecting duct

Renal sodium reabsorption depends on the activity of the Na⁺,K⁺-ATPase α/β heterodimer. Four α (α_1–4) and 3 β (β_1–3) subunit isoforms have been described. It is accepted that renal tubule cells express α₁/β₁ dimers. Aldosterone stimulates Na⁺,K⁺-ATPase activity and may modulate α₁/β₁ expression. However, some studies suggest the presence of β₃ in the kidney. We hypothesized that the β₃ isoform of the Na⁺,K⁺-ATPase is expressed in tubular cells of the distal nephron, and modulated by mineralocorticoids. We found that β₃ is highly expressed in collecting duct of rodents, and that mineralocorticoids decreased the expression of β₃. Thus, we describe a novel molecular mechanism of sodium pump modulation that may contribute to the effects of mineralocorticoids on sodium reabsorption.     

Chromosomal locations of the gene coding for the CD3 (T3) γ subunit of the human and mouse CD3/T-cell antigen receptor complexes

The gene coding for the M _r 26000 chain of the human CD3 (T3) antigen/T-cell antigen receptor complex was mapped to chromosome band 11q23 by using a cDNA clone (pJ6T3 -2), by in situ hybridization to metaphase chromosomes and by Southern blot analysis of a panel of human-rodent somatic cell hybrids. The mouse homolog, here termed Cdg-3, was mapped to chromosome 9 using the mouse cDNA clone pB10.AT3 -1 and a panel of mouse-hamster somatic cell hybrids. Similar locations for the CD3 genes have been described previously. Thus, the corporate results indicate that the CD3 and genes have remained together since they duplicated about 200 million years ago.     

Expression of the Clustered NeuAcα2–3Galβ O-Glycan Determines the Cell Differentiation State of the Cells

Human embryonic stem cells (hESCs) are pluripotent stem cells from early embryos, and their self-renewal capacity depends on the sustained expression of hESC-specific molecules and the suppressed expression of differentiation-associated genes. To discover novel molecules expressed on hESCs, we generated a panel of monoclonal antibodies against undifferentiated hESCs and evaluated their ability to mark cancer cells, as well as hESCs. MAb7 recognized undifferentiated hESCs and showed a diffuse band with molecular mass of >239 kDa in the lysates of hESCs. Although some amniotic epithelial cells expressed MAb7 antigen, its expression was barely detected in normal human keratinocytes, fibroblasts, or endothelial cells. The expression of MAb7 antigen was observed only in pancreatic and gastric cancer cells, and its levels were elevated in metastatic and poorly differentiated cancer cell lines. Analyses of MAb7 antigen suggested that the clustered NeuAcα2–3Galβ O-linked oligosaccharides on DMBT1 (deleted in malignant brain tumors 1) were critical for MAb7 binding in cancer cells. Although features of MAb7 epitope were similar with those of TRA-1–60, distribution of MAb7 antigen in cancer cells was different from that of TRA-1–60 antigen. Exposure of a histone deacetylase inhibitor to differentiated gastric cancer MKN74 cells evoked the expression of MAb7 antigen, whereas DMBT1 expression remained unchanged. Cell sorting followed by DNA microarray analyses identified the down-regulated genes responsible for the biosynthesis of MAb7 antigen in MKN74 cells. In addition, treatment of metastatic pancreatic cancer cells with MAb7 significantly abrogated the adhesion to endothelial cells. These results raised the possibility that MAb7 epitope is a novel marker for undifferentiated cells such as hESCs and cancer stem-like cells and plays a possible role in the undifferentiated cells.     

Peptide analogues of 1811–1818 loop of the A3 subunit of the light chain A3-C1–C2 of FVIII of blood coagulation: biological evaluation

Factor VIII, the plasma protein deficient or defective in individuals with hemophilia A, is a critical member of the blood coagulation cascade. Recent studies have identified the FVIII light chain region Glu1811-Lys1818 as being involved in FIXa binding and in the assembly of the FX-activating FIXaz–FVIIIa complex. Based on this, a series of 12 peptides, analogues of the 1811–1818 loop of the A3 subunit of the light chain A3-C1–C2 of FVIIIa, were synthesized and evaluated for their anticoagulant activity. Only peptide Ac-ETKTYFWK-NH₂ showed significant anticoagulant activity by inhibiting about 40% factor VIII at a concentration of 0.43 mM. It also showed a prolongation of activated partial thromboplastin time of 6.1 s, whereas its effect on prothrombin time measurements was meaningless. All the other peptides did not show any measurable effect at the concentration of 0.43 mM. These findings are encouraging though further investigation of the effect of this active peptide in different biological settings is needed in order to evaluate its possible clinical applications.     

The influence of inhibiting or stimulating the expression of the α3 subunit of the nicotinic receptor in SH-SY5Y cells on levels of amyloid-β peptide and β-secretase

To examine the effects of the α3 subunit of the nicotinic acetylcholine receptor (nAChR) on the expression of β-secretase and the concomitant level of amyloid-β (Aβ), SH-SY5Y neuroblastoma cells were either transfected with small interference RNAs (siRNAs) specifically targeting this subunit or exposed to nicotine. The levels of α3 nAChR mRNA and protein, as well as the corresponding levels of BACE1 (which cleaves the β-site of APP) and BACE2 (cleaving in the Aβ domain) were determined by real-time PCR and Western blotting, respectively. The levels of Aβ_1–42 in culture media were determined by an Elisa procedure.     

A reconstituted cell-free system for the specific transfer of steroid–receptor complexes into nuclear chromatin isolated from rat ventral prostate gland

1. A system has been developed for the specific transfer of [(3)H]dihydrotestosterone-receptor complexes into prostatic chromatin in vitro. 2. Under optimum conditions the overall transfer of [(3)H]dihydrotestosterone into purified chromatin in this reconstituted system is entirely consistent with the results obtained in whole tissue both in vivo and in vitro. 3. The transfer of [(3)H]dihydrotestosterone into chromatin is tissue-specific and maximal into chromatin isolated from androgen-dependent tissues. 4. The tissue specificity is maintained at two levels: first, in the presence of specific cytoplasmic androgen-receptor proteins; secondly, by the nature and composition of the chromatin itself. 5. Evidence is presented that androgenic steroids in vivo may maintain the tissue-specific nature of chromatin in androgen-dependent tissues by the selective induction of nuclear protein synthesis. 6. The relevance of these findings to the mechanism of action of androgenic steroids is discussed.     

High-level expression of a recombinant fragment of human fibronectin containing the Cell I–Hep II–IIICS71 domain in Escherichia coli as a soluble protein

Fibronectin (FN) is a major matrix protein that is involved in multiple processes. Its Cell I–Hep II domain is potentially useful in tumor therapy. Here, a recombinant fragment of FN with the Cell I–Hep II-IIICS71 domain, CH/71, was expressed in Escherichia coli. The CH/71 fusion protein consists of Cell I–Hep II domain and 19th to 89th amino acids of IIICS domain of FN. The expression level of CH/71 in E. coli was very high after induction with IPTG. Furthermore, CH/71 protein was largely found in the soluble fraction. It was readily purified by one-step heparin–agarose affinity chromatograph. The ability of CH/71 binding cells was about 8-fold of that of Cell I–Hep II domain FN.     

Effect of serum type and concentration on the expression ofβ-galactosidase in recombinant vaccinia virus infected HeLa S3 cells

The effect of serum type and concentration on recombinant protein expression in vaccinia virus infected HeLa S3 cells was studied in both static and suspension culture. A model heterologous protein,-galactosidase (-gal), was used. Calf and horse sera in the range of 0.5–10%(v/v) were investigated. In static culture, the calf serum concentration did not show any significant influence on the -gal production which was almost completed within 24h postinfection (pi). Higher horse serum concentration, on the other hand, resulted in higher -gal concentration which continued to increase until 48 h pi. Total -gal concentrations in 0.5% calf serum at 24 h pi and 10% horse serum at 48 h pi were 2.2±0.7 and 2.2±0.1 IU/ml, respectively. In suspension culture, both sera showed their respective effects on the -gal production similar to those observed in static culture, indicating that the cultivation method had little influence on -gal production. Accordingly, the use of 0.5% calf serum after virus infection in recommended for economical -gal production.     

p38 MAP kinase–dependent phosphorylation of the Gp78 E3 ubiquitin ligase controls ER–mitochondria association and mitochondria motility

Gp78 is an ERAD-associated E3 ubiquitin ligase that induces degradation of the mitofusin mitochondrial fusion proteins and mitochondrial fission. Gp78 is localized throughout the ER; however, the anti-Gp78 3F3A monoclonal antibody (mAb) recognizes Gp78 selectively in mitochondria-associated ER domains. Epitope mapping localized the epitope of 3F3A and a commercial anti-Gp78 mAb to an 8–amino acid motif (533–541) in mouse Gp78 isoform 2 that forms part of a highly conserved 41–amino acid region containing 14-3-3– and WW-binding domains and a p38 MAP kinase (p38 MAPK) consensus site on Ser-538 (S538). 3F3A binds selectively to nonphosphorylated S538 Gp78. Using 3F3A as a reporter, we induced Gp78 S538 phosphorylation by serum starvation and showed it to be mediated by p38 MAPK. Mass spectroscopy analysis of Gp78 phosphopeptides confirmed S538 as a major p38 MAPK phosphorylation site on Gp78. Gp78 S538 phosphorylation limited its ability to induce mitochondrial fission and degrade MFN1 and MFN2 but did not affect in vitro Gp78 ubiquitin E3 ligase activity. Phosphomimetic Gp78 S538D mutation prevented Gp78 promotion of ER–mitochondria interaction, and SB203580 inhibition of p38 MAPK increased ER–mitochondria association. p38 MAPK phosphorylation of Gp78 S538 therefore regulates Gp78-dependent ER–mitochondria association and mitochondria motility.     

Phosphorylation and dephosphorylation of the regulatory subunit of cyclic 3′,5′-monophosphate-dependent protein kinase (type II) in vivo and in vitro

A phosphorylated regulatory subunit of cyclic AMP-dependent protein kinase (type II) was purified to homogeneity from inorganic [³²P]phosphate-injected rats.A new method of measuring the phosphorylation reaction was developed. It was found that this regulatory subunit was phosphorylated in cells and comprised 60, 82 and 55% of the total regulatory subunit in brain, heart and liver cytosol fractions from rats, respectively.Dephosphorylation was stimulated by cyclic nucleotides. The K_a values for cyclic AMP and cyclic IMP were 0.30 and 1.0 μM, respectively. Purified phosphoprotein phosphatase could dephosphorylate the regulatory subunit and this reaction was also stimulated by cyclic nucleotides with similar K_a values. The inhibitors of phosphoprotein phosphatase, NaF and ZnCl₂, protected against dephosphorylation unless ADP or cyclic AMP were present.     

Oral–aboral identity displayed in the expression of HpHox3 and HpHox11/13 in the adult rudiment of the sea urchin Holopneustes purpurescens

Hox genes are noted for their roles in specifying axial identity in bilateral forms. In the radial echinoderms, the axis whose identity Hox genes might specify remains unclear. From the expression of Hox genes in the development of the sea urchin Holopneustes purpurescens reported here and that reported previously, we clarify the axis that might be specified by Hox genes in echinoderms. The expression of HpHox11/13 here is described at three developmental stages. The expression is around the rim of the blastopore in gastrulae, in the archenteron wall and adjacent mesoderm in early vestibula larvae, and in a patch of mesoderm close to the archenteron wall in later vestibula larvae. The retained expression of HpHox11/13 in the patch of mesoderm in the later vestibula larvae is, we suggest, indicative of a posterior or an aboral growth zone. The expression of HpHox3 at the echinoid-rudiment stage, in contrast, is in oral mesoderm beneath the epineural folds, concentrated in sites where the first three adult spines form. With the expression of HpHox5 and HpHox11/13 reported previously, the expressions here support the role of Hox genes in specifying oral–aboral identity in echinoderms. How such specification and a posterior growth zone add support to a concept of the structural homology between echinoderms and chordates is discussed.     

Hsa_circ_0069094 positively regulates the expression of oncogenic ZNF217 by competitively targeting miR-758–3p to promote the development of breast cancer

To investigate the functions and potential mechanisms of hsa_circ_0069094 in this cancer. The expression of hsa_circ_0069094, zinc finger protein 217 (ZNF217) and microRNA-758–3p (miR-758–3p) was detected by quantitative polymerase chain reaction (qPCR), and the protein level of ZNF217 was detected by western blot. Cell proliferation was assessed using cell counting kit-8 (CCK-8) assay and colony formation assay. Cell cycle progression and cell apoptosis were determined using flow cytometry assay. Cell invasion and cell migration were monitored using transwell assay and wound healing assay. The protein levels of apoptosis-related proteins were quantified by western blot. The putative relationship between miR-758–3p and hsa_circ_0069094 and ZNF217 was confirmed using dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Xenograft model was constructed in mice to explore the role of hsa_circ_0069094 on solid tumor growth.Hsa_circ_0069094 and ZNF217 were highly expressed, while miR-758–3p was poorly expressed in tissues and cells of breast cancer. Hsa_circ_0069094 knockdown or ZNF217 knockdown inhibited cell proliferation, invasion and migration and induced cell apoptosis and cell cycle arrest in breast cancer cells. The inhibitory effects of hsa_circ_0069094 knockdown on cell malignant behaviors were abolished by ZNF217 overexpression. Hsa_circ_0069094 competed with ZNF217 for the binding site of miR-758–3p, and hsa_circ_0069094 positively regulated ZNF217 expression by competitively binding to miR-758–3p. Hsa_circ_0069094 knockdown also blocked solid tumor growth in mice. Collectively, Hsa_circ_0069094 played oncogenic effects in breast cancer by activating the expression of ZNF217 via competitively binding to miR-758–3p, which might be a novel strategy for breast cancer suppression.