Unipolar brush cells (UBCs) are excitatory interneurons with their somata located in the granular layer. Recently, T-brain factor 2 (Tbr2) was shown to be expressed in a subset of UBCs in mouse cerebellum. Scrambler mice exhibit severe cerebellum abnormalities, including the failure of embryonic Purkinje cell dispersal and a complete absence of foliation due to a mutation in the disabled-1 adaptor protein. Since most UBC markers are expressed postnatally, it has proven difficult to identify the relationship between developing Purkinje cell clusters and migrating UBCs. Because scrambler mice closely mimic normal embryonic day 18 cerebellum, we examined whether Tbr2-positive UBCs are associated with Purkinje cell cluster markers such as zebrin II, which is the most studied compartmentation marker in the cerebellum. We investigated the distribution of Tbr2-positive UBCs in this mutant by using anti-Tbr2 immunocytochemistry. The data revealed that Tbr2 immunoreactivity was exclusively present in the nucleus of UBCs in scrambler cerebellum. Based on expression data, a Tbr2-positive UBC map was constructed. In addition, Tbr2-positive UBCs are found associated with ectopic zebrin II-immunoreactive Purkinje cell clusters in scrambler cerebellum. These data suggest that UBCs use Purkinje cell compartmentation to migrate into their final position through interactions with the embryonic array of specific Purkinje cell subtypes.
Animals ; Cell Compartmentation ; Cerebellum ; Hydrazines ; Immunohistochemistry ; Interneurons ; Mice ; Nerve Tissue Proteins

This experimental research was aimed to establish an optimum system of enucleation, purification and identification for preparing the cytoplasts of suspension culture cells in order to undertake cell recombination. Human leukemia HL-60 cells in suspension culture were purified by 42% Percoll density gradient centrifugation and low-speed centrifugation at 1 500r/min, respectively. The purified HL-60 cells were treated with cytochalasin B (CB) alone or combined with colcchicine and enucleated by isopycnic gradient centrifugation on 50% Percoll at 25 degrees C and 34 degrees C, respectively. Cytoplasts made from HL-60 cells were purified through gradient centrifugation by 37%, 38% and 40% Percoll, respectively. The final cytoplasts were identified by Wright-Giemsa staining and 4,6-diamidino-2-phenylidole dihydrochloride (DAPI)/5, 6-carboxyflu-orescein diacetate succinimidyl ester (CFSE) double-staining. The phenotype and mitochondrial membrane potential of HL-60 cytoplasts were analyzed by flow cytometry. The results indicated that the enucleation ratio of HL-60 cells induced by CB combined with colcchicine was up to 91. 98% +/-4. 29%, which was significantly higher than that in CB alone group (74. 95% +/- 3. 02%)(P<0. 01). The rates of enucleation and cytoplast with diameter over 5min in 34 degrees C group were higher than those in 25 degrees C group (all P<0. 01). The cytoplast purities were (95.43 +/- 0. 59)% in 38% Percoll groups,which were higher than those of 40% Percoll (P<0. 05). Nucleus and caryoplasm could be clearly distinguished by DAPI and CFSE double labeling. The results further showed that the phenotype of HL-60 cytoplasts had no significant change, and the activity of the cytoplasts was above 80% within 12h. It is concluded that enucleation throuth density gradient centrifugation on 50% Percoll mediated by CB combined with colcchicine, 38%Percoll of purification followed by DAPI/CFSE double labeling and MMP detection is an optimum scheme for preparation and identification of cytoplast from suspension culture cells.
Cell Compartmentation ; Cell Nucleus ; Cell Separation ; Centrifugation, Density Gradient ; Colchicine ; analogs & derivatives ; pharmacology ; Cytochalasin B ; pharmacology ; Cytoplasm ; HL-60 Cells ; Humans

BACKGROUNDThe aim of this study was to identify the subnuclear distribution pattern of human orphan nuclear receptor steroidogenic factor 1 (SF-1) in living cells with and without the activation of protein kinase A (PKA) signal pathway, and thus try to explain the unknown mechanism by which PKA potentiates SF-1 transactivation.

METHODSFull-length cDNAs of wild type and a naturally occurring mutant (G35E) human SF-1 were cloned and fused with green fluorescent protein (GFP). Subcellular distribution pattern of human SF-1 in living cells, whose PKA signaling was either activated or not, was studied by laser confocal microscopy after the validity of the gene sequence was confirmed.

RESULTSThe transactivation ability of the GFP-SF-1 chimeric protein was highly conserved. Wild type human SF-1 diffused homogeneously within the nuclei of cells when PKA was not active, and converged to clear foci when PKA was activated. Mutant SF-1 diffused within the nuclei even in the presence of PKA activation, surprisingly aggregating as fluorescent dots inside the nucleoli, a phenomenon not altered by PKA.

CONCLUSIONSActivation of PKA causes wild type, but not mutant SF-1 to alter its subnuclear distribution pattern to a transactivationally active form (foci formation). This finding may throw new light on the mechanism by which PKA activates the orphan nuclear receptor.

Cell Compartmentation ; Cell Nucleus ; chemistry ; Cells, Cultured ; Colforsin ; pharmacology ; Cyclic AMP-Dependent Protein Kinases ; physiology ; DNA-Binding Proteins ; analysis ; Enzyme Activation ; Female ; Fushi Tarazu Transcription Factors ; Homeodomain Proteins ; Humans ; Microscopy, Confocal ; Receptors, Cytoplasmic and Nuclear ; Steroidogenic Factor 1 ; Transcription Factors ; analysis ; Transcriptional Activation

OBJECTIVESThe hepatitis B virus core protein has been found in nuclei, cytoplasm, or both of hepatocytes transfected with HBV DNA. It is still unclear whether intact core particles could pass through nuclear pores and what could be the mechanism regulating the subcellular localization of the core protein. This study on the distribution of core protein in hepatocytes and its translocation has a potential advantage to learn more about the HBV life cycle.

METHODSDimethyl sulphoxide (DMSO, 2%), which effects hepatic differentiation, and/or 1 micro mol/L heteroaryldihydropyrimidine Bay41-4109, which interferes with the assembly of core particles, were added into HepG2.2.15 cell culture system for 4 days. The hepatitis B virus core antigen (HBcAg) and hepatitis B virus surface antigen (HBsAg) were stained with fluorescent immunocytochemistry and then observed under a confocal microscope. HBcAg in cytoplasm and nuclei were respectively extracted and analyzed using Western blot. HBV covalently closed circular DNA (cccDNA) was detected by using selective PCR method.

RESULTSThe HBcAg was mostly expressed in the cytoplasm and weak signals of cccDNA were detected in the control HepG2.2.15 cells. After DMSO treatment, the expression of HBcAg in cytoplasm was increased about 2.5-fold; the expression of HBcAg and cccDNA in nuclei also increased. With the use of Bay41-4109, the signal of HBcAg in cytoplasm decreased 2/3, but it increased in the nuclei, and cccDNA decreased in the nuclei. When the HepG2.2.15 cells were treated both with DMSO and Bay41-4109, cord-liked distribution of HBsAg was observed in the cytoplasm. HBcAg in cytoplasm was decreased 1/2 but the HBcAg in the nuclei increased about 5-fold, whereas the cccDNA was almost negative.

CONCLUSIONIn HepG2.2.15 cells, the core protein is mainly assembled as a formation of core particles in the cytoplasm and they are blocked by the nuclear membrane. Bay41-4109 interferes with the assembly of core particles and the dissociated core proteins are able to enter the nuclei. DMSO promotes the nuclear entry of core protein/core particles and facilitates the formation of cccDNA.

Chromosome Positioning ; Dimethyl Sulfoxide ; pharmacology ; Hep G2 Cells ; Hepatitis B Core Antigens ; metabolism ; Hepatitis B virus ; physiology ; Humans ; Neoplasm Metastasis ; Pyridines ; pharmacology ; Pyrimidines ; pharmacology ; Viral Core Proteins ; metabolism ; Virus Assembly