In vivo two-photon calcium imaging of cortical neurons in neonatal mice.

In vivo calcium imaging is essential to elucidate unique synchronous activities observed in the developing brain. Here, we present a protocol to image and analyze activity patterns in neonatal mouse neocortex in a single-cell level. We describe steps for in utero electroporation, cranial window surgery, two-photon imaging, and activity correlation analysis. This protocol facilitates the understanding of neuronal activities and activity-dependent circuit formation during development. For complete details on the use and execution of this protocol, please refer to Mizuno et al. (2014),1 Mizuno et al. (2018a),2 and Mizuno et al. (2018b).3.

Ras GTPase-activating proteins control neuronal circuit development in barrel cortex layer 4.

The cerebral cortex comprises a complex and exquisite network of neuronal circuits that is formed during development. To explore the molecular mechanisms involved in cortical circuit formation, the tactile somatosensory pathway that connects the whiskers and cortex of rodents is a useful model. Here, we analyzed the roles of Ras GTPase-activating proteins (RasGAPs) in the circuit formation in the somatosensory cortex layer 4 (L4). We suppressed the function of RasGAPs in L4 neurons using Supernova RNAi, a plasmid vector-based sparse cell gene knockdown (KD) system. The results showed disrupted dendritic pattern formation of L4 spiny stellate neurons on the barrel edge by RasGAP KD. Furthermore, the number of presynaptic boutons on L4 neurons was reduced by RasGAP KD. These results demonstrate the essential roles of RasGAPs in circuit formation in the cerebral cortex and imply that developmental changes in dendrites and synapses in RasGAP KD neurons may be related to cognitive disabilities in RasGAP-deficient individuals, such as patients with neurofibromatosis type 1.

NMDA Receptor Enhances Correlation of Spontaneous Activity in Neonatal Barrel Cortex.

Correlated spontaneous activity plays critical role in the organization of neocortical circuits during development. However, cortical mechanisms regulating activity correlation are still elusive. In this study, using two-photon calcium imaging of the barrel cortex layer 4 (L4) in living neonatal mice, we found that NMDA receptors (NMDARs) in L4 neurons are important for enhancement of spontaneous activity correlation. Disruption of GluN1 (Grin1), an obligatory NMDAR subunit, in a sparse population of L4 neurons reduced activity correlation between GluN1 knock-out (GluN1KO) neuron pairs within a barrel. This reduction in activity correlation was even detected in L4 neuron pairs in neighboring barrels and most evident when either or both of neurons are located on the barrel edge. Our results provide evidence for the involvement of L4 neuron NMDARs in spatial organization of the spontaneous firing activity of L4 neurons in the neonatal barrel cortex.SIGNIFICANCE STATEMENT Precise wiring of the thalamocortical circuits is necessary for proper sensory information processing, and thalamus-derived correlated spontaneous activity is important for thalamocortical circuit formation. The molecular mechanisms involved in the correlated activity transfer from the thalamus to the neocortex are largely unknown. In vivo two-photon calcium imaging of the neonatal barrel cortex revealed that correlated spontaneous activity between layer four neurons is reduced by mosaic knock-out (KO) of the NMDA receptor (NMDAR) obligatory subunit GluN1. Our results suggest that the function of NMDARs in layer four neurons is necessary for the communication between presynaptic and postsynaptic partners during thalamocortical circuit formation.

Involvement of transcription factor Ets-1 in the expression of the α3 integrin subunit gene.

The α3ß1 integrin is an adhesion receptor for extracellular matrix proteins, and plays crucial roles in cell motility, proliferation, and differentiation. The aberrant expression of this adhesion molecule on tumor cells is frequently associated with their malignant behaviors. We previously reported that the Ets transcription factor-binding consensus sequence 133 bp upstream of the mouse α3 integrin gene is an important element for its expression in various tumor cell lines. In the present study, we attempted to identify a transcription factor bound to the Ets-consensus sequence, and found that Ets-1 bound to this sequence in an electrophoretic mobility shift assay, chromatin immunoprecipitation assay, and pull-down assay with a tandem repeat of the sequence as adsorbent. We next examined the role of Ets-1 in α3 integrin gene expression by use of a luciferase assay with a reporter plasmid containing the 5'-flanking region of the α3 integrin gene. Cotransfection of HEK293T cells with an Ets-1 expression construct and the reporter plasmid increased luciferase activity. By contrast, transfection of HT1080 cells (high α3 integrin expresser) with a dominant-negative mutant of Ets-1 decreased luciferase activity. Overexpression of Ets-1 in HepG2 hepatocellular carcinoma cells (low α3 integrin expresser) upregulated α3 integrin expression as assessed by immunoprecipitation. Finally, the induction of α3 integrin gene expression in HepG2 cells after transforming growth factor-ß1 treatment was abrogated by the dominant-negative mutant of Ets-1. These results suggest that Ets-1 is involved in transcriptional activation of the α3 integrin gene through its binding to the Ets-consensus sequence at -133 bp.

Polyhydroxylated fullerene C60(OH)44 suppresses intracellular lipid accumulation together with repression of intracellular superoxide anion radicals and subsequent PPARγ2 expression during spontaneous differentiation of OP9 preadipocytes into adipocytes.

Reactive oxygen species has been suggested to be one of the key factors associated with the development of obesity. During spontaneous differentiation of mouse stromal preadipocytes OP9 into adipocytes, intracellular superoxide anion radicals (O (2) (-.) ) level markedly increases and is accompanied by a significant elevation of intracellular lipid accumulation. This differentiation-dependent increase in intracellular O (2) (-.) level positively correlated with the intracellular augmentation of the lipid level. Super-highly hydroxylated fullerene (SHH-F; C(60)(OH)(44)), a novel polyhydroxylated fullerene derivative, quenched intracellular O (2) (-.) , and lipid accumulation to 38.7 and 42.7 % of that in the control, respectively. By thin-layer chromatographic analysis of extracted cellular lipid components, SHH-F clearly decreased the triglycerides ratio in the whole lipid droplet fraction, but scarcely influenced other lipids components. PPARγ2 expression, which plays a key role in regulating adipogenic differentiation, was significantly suppressed by SHH-F at the late stage of differentiation, with unaltered PPARγ1 expression. The intracellular superoxide anion radical augmentation preceded expression of PPARγ2, strongly suggesting that the primary O (2) (-.) generation was closely associated with lipid accumulation and subsequent PPARγ2 induction. These results indicate that SHH-F suppresses intracellular lipid accumulation, particularly in lipid droplets, and decreases O (2) (-.) level and subsequent PPARγ2 upregulation during spontaneous differentiation of OP9 preadipocytes into adipocytes.

Monocyte differentiation induced by co-culture with tumor cells involves RGD-dependent cell adhesion to extracellular matrix.

Macrophages that infiltrate tumor tissues, or tumor-associated macrophages (TAMs), affect the malignant behaviors of tumor cells. In this study, we attempted to induce monocytes to differentiate into TAM-like cells producing matrix metalloproteinases (MMPs) by co-culture with tumor cells. When human monocytes were co-cultured for 3-7 days with tumor cell lines, monocytes differentiated to produce MMP-9, accompanied by morphological changes. The in vitro cell invasion of MKN1 human gastric carcinoma cells into Matrigel membranes was promoted in the presence of differentiated monocytes, and the enhancement of cell invasion by differentiated monocytes was correlated with their MMP-9 productivity. The addition of an RGD (Arg-Gly-Asp) peptide to the culture significantly inhibited monocyte differentiation. The MMP-9 production from monocytes was diminished by the depletion of fibronectin from the conditioned media with gelatin-Sepharose, and potentiated by culturing them in fibronectin-coated plates. These results suggest that cell adhesion to the extracellular matrix plays a crucial role in monocyte differentiation into TAM-like cells.

Super-highly hydroxylated fullerene derivative protects human keratinocytes from UV-induced cell injuries together with the decreases in intracellular ROS generation and DNA damages.

Polyhydroxylated fullerenes (fullerenols: C(60)(OH)(n)) are known as the major water-soluble fullerene derivatives which possess particular significance as free radical scavengers or antioxidants in biological systems. Recently, the novel polyhydroxylated fullerene (C(60) (OH)(44)·8H(2)O: SHH-F) was successfully synthesized. In the present study, we investigated the radical-scavenging effects and cytoprotective effects of three types of fullerenols (C(60)(OH)(6-12): LH-F, C(60) (OH)(32-34)·7H(2)O: HH-F, and C(60) (OH)(44)·8H(2)O: SHH-F) on UV-irradiation-induced cell injuries. HH-F and SHH-F exerted hydroxyl-radical scavenging activities as shown by DMPO-spin trap/ESR method, more markedly than LH-F. UVA or UVB irradiation-induced injuries in human skin keratinocytes HaCaT were significantly suppressed by HH-F and SHH-F, but scarcely by LF-H. The cytoprotective effects of SHH-F had a tendency to be superior to that of HH-F. And the cytoprotective effects of SHH-F against UVB-induced injuries were more effective than those of UVA. Irradiation with UVB to HaCaT cells was shown to cause rapid increases in cell-injury-associated symptoms such as intracellular oxidative stress levels, the formation of cyclobutane pyrimidine dimers and chromatin condensation, all of which were repressed by SHH-F. Thus, UVB-induced diverse harmful effects could be prevented by SHH-F, which was suggested to exert the cytoprotective effects through intracellular reactive oxygen species-scavenging in the keratinocytes.

Development of an enzyme-linked immunosorbent assay for measurement of rat pulmonary surfactant protein D using monoclonal antibodies.

Surfactant protein D (SP-D) has been used as a biomarker of lung inflammation. In rat, several types of enzyme-linked immunosorbent assay (ELISA) using polyclonal antibodies have been reported. The purpose of this study was the development of a sensitive ELISA for rat SP-D using monoclonal antibodies. The authors developed a sandwich ELISA using monoclonal antibodies that were obtained by immunizing with purified rat SP-D. The ELISA was evaluated by performance tests. Furthermore, concentrations of serum SP-D were measured in normal control and bleomycin-treated rats. The working range of ELISA was between 0.47 and 30 ng/mL. Different concentrations of added SP-D were recovered, between 94.1% and 102.8%. Serum SP-D levels of bleomycin-treated rats were significantly higher than those of normal rats. In conclusion, this newly developed ELISA for rat SP-D using monoclonal antibodies is applicable for research on the mechanism and therapy of lung injury.

Novel polyhydroxylated fullerene suppresses intracellular oxidative stress together with repression of intracellular lipid accumulation during the differentiation of OP9 preadipocytes into adipocytes.

Along with differentiation of mouse stromal preadipocytes OP9 into adipocytes, intracellular ROS, especially superoxide anion radicals detected by NBT reduction assay, were found to appreciably increase, mainly in cytoplasmic area, parallelling with increases in intracellular lipid-droplet accumulation, whereas undifferentiated OP9 cells kept lower levels of ROS and lipid-droplets. beta-Carotene bleaching assay showed that super-highly hydroxylated fullerene (SHH-F; C(60) (OH)(44)) exerted higher antioxidant ability than highly hydroxylated fullerene (HH-F; C(60) (OH)(32-34)) or lowly hydroxylated fullerene (LH-F; C(60) (OH)(6-12)). Differentiation-dependent lipid-droplet accumulation was suppressed by SHH-F or HH-F more efficiently than LH-F. Furthermore, SHH-F significantly repressed intracellular ROS generation accompanied by adipocyte differentiation. Thus, lipid-droplet accumulation was shown to positively correlate with ROS upon the differentiation of OP9 preadipocytes into adipocytes and SHH-F significantly suppressed intracellular ROS together with repression of intracellular lipid accumulation.

Potentiation of cell invasion and matrix metalloproteinase production by alpha3beta1 integrin-mediated adhesion of gastric carcinoma cells to laminin-5.

We previously reported that the adhesion of gastric carcinoma cells to the peritoneum mediated by the alpha3beta1 integrin-laminin interaction is a key step in the initial process of peritoneal metastatic dissemination. Carcinoma cells subsequently invade through the intercellular gaps of mesothelial linings. In this study, we examined the role of the interaction of carcinoma cells with laminin-5, which is a major component of submesothelial basement membranes and serves as a high-affinity ligand for alpha3beta1 integrin, in carcinoma cell invasion. Human gastric carcinoma cell lines (MKN1, GT3TKB, and NUGC-4) adhered in an alpha3beta1 integrin-dependent manner to the extracellular matrix deposited by peritoneal mesothelial cells. An in vitro invasion assay using the Boyden chamber system revealed that MKN1 cell migration through the membranes increased when the membranes were coated with matrices produced by mesothelial cells or with laminin-5-containing Matrigel as compared to Matrigel alone. The cell migration promoted by laminin-5-containing Matrigel was inhibited by the presence of anti-alpha3 integrin antibody. When MKN1 cells were cultured in a laminin-5-coated plate, these cells were promoted to produce matrix metalloproteinase (MMP)-9, as assessed by gelatin zymography, enzyme-linked immunosorbent assay, and reverse transcription-polymerase chain reaction. These results suggest that the production of MMP-9 by MKN1 cells was potentiated by the alpha3beta1 integrin-laminin-5 interaction, which facilitated their invasion via degradation of the matrix.

Changes in adhesive and migratory characteristics of hepatocellular carcinoma (HCC) cells induced by expression of alpha3beta1 integrin.

The invasive and metastatic potentials of hepatocellular carcinoma are positively correlated with the expression level of alpha3beta1 integrin, a high-affinity adhesion receptor for laminin isoforms including laminin-5. In this study, we investigated changes in the adhesive and invasive behaviors of human HCC HepG2 cells after transfection with cDNA for alpha3 integrin in order to elucidate the direct involvement of this integrin in these cellular processes. We introduced cDNA for splice variants of alpha3 integrin (alpha3A and alpha3B) into the cells, and selected two transfectant clones (HepG2-3A and HepG2-3B), which express the alpha3A and alpha3B integrins, respectively. Both transfectant cells adhered almost equally to laminin-5-coated plates in an alpha3 integrin-dependent manner, indicating that transfected alpha3Abeta1 and alpha3Bbeta1 integrins were functionally active in these cells. The migratory and invasive potentials of the transfectant cells were assessed by scratch wound assay and in vitro chemoinvasion assay. The results demonstrated that the migration of HepG2-3A and HepG2-3B cells but not of mock transfectant (HepG2-M) cells was stimulated on the plates coated with laminin-5. Furthermore, HepG2-3A and HepG2-3B cells were found to be more invasive into laminin-5-containing matrices than were HepG2-M cells. These results strongly suggest that enhanced expression of alpha3beta1 integrin on HCC cells is directly involved in their malignant phenotypes such as invasion and metastasis.

Characterization of the promoter for the alpha3 integrin gene in various tumor cell lines: roles of the Ets- and Sp-family of transcription factors.

The alpha3beta1 integrin is an adhesion receptor for extracellular matrix proteins, including laminin isoforms, and plays crucial roles in the organization of epithelial and endothelial tissues. The aberrant expression of this adhesion molecule on tumor cells is associated with their invasive and metastatic potentials. In the present study, we analyzed the elements essential for alpha3 integrin gene expression in various tumor cell lines with different tissue origins by luciferase assay. An approximately 0.3 kb fragment of the 5'-flanking region of the mouse alpha3 integrin gene (-260/+84, relative to the major transcription start site) showed strong promoter activity in all six examined tumor cell lines. However, we found that these cell lines could be divided into two groups according to the level of dependency on the putative Ets-transcription factor binding motif located at -133. This motif was previously shown to be crucial for alpha3 integrin expression in MKN1 gastric carcinoma cells. The gene expression in one group of cell lines was upregulated mainly by the Ets motif, whereas that in the other group was less dependent on the Ets motif. We then postulated that additional regulatory elements were responsible for the expression of alpha3 integrin, and found that a GC-rich motif at -69 was another important element. An electrophoretic mobility shift assay using specific antibodies and a Western blot analysis of nuclear proteins revealed that the Sp3-transcription factor bound to this GC-rich motif. These results suggest that the Sp3 and Ets transcription factors cooperatively regulate alpha3 integrin gene expression and that the contribution of each element depends on the type of tumor cells.

Both HDL3 and HDL2 exert a powerful anti-oxidative and protective effect against acceleration of oxidative modification of LDL by ascorbic acid.

The objective of the present study was to establish whether high-density lipoprotein 3 (HDL3) or high-density lipoprotein 2 (HDL2) might show an anti-oxidative effect on the acceleration of the oxidative modification of low-density lipoprotein (LDL) by ascorbic acid from measurement of the agarose gel electrophoretic mobility of LDL. LDL was incubated without adding transitional-metal ions for 48 or 96 h in phosphate-buffered saline (PBS) alone, with ascorbic acid (20 microg/mL), or with both ascorbic acid (20 microg/mL) and HDL3 (200 microg protein/mL). The LDL autoxidation occurred in PBS alone. Although ascorbic acid significantly suppressed oxidative modification of LDL after incubation for 48 h, the opposite was true after 96 h. However, since the anti-oxidative ability of HDL2 shows a weaker tendency than that of HDL3, both HDL3 and HDL2 significantly inhibited this acceleration of oxidative modification of LDL by ascorbic acid as assessed by electrophoretic mobility. If there is an augmented oxidative modification of LDL due to ascorbic acid in vivo, HDL3 or HDL2 may thus have an important role in inhibiting this ascorbic acid-accelerated oxidation of LDL.

Transforming growth factor-beta1 upregulates transcription of alpha3 integrin gene in hepatocellular carcinoma cells via Ets-transcription factor-binding motif in the promoter region.

The invasive and metastatic potentials of hepatocellular carcinoma (HCC) are positively correlated with the expression level of alpha3beta1 integrin, a high-affinity adhesion receptor for laminin isoforms. Transforming growth factor (TGF)-beta1 stimulates non-invasive HCC cells to acquire invasive phenotypes in association with the enhanced expression of alpha3 integrin. In this study, we investigated the molecular mechanism underlying the upregulation of alpha3beta1 integrin by TGF-beta1 in non-invasive HepG2 HCC cells. The treatment of HepG2 cells with TGF-beta1 induced the expression of alpha3 integrin and potentiated these cells to adhere to laminin-5 and to migrate through laminin-5-coated membranes. The promoter activity was measured by luciferase assay with a series of deletion constructs of the 5'-flanking region of the mouse alpha3 integrin gene, and the results showed that the -260/-119 region (relative to the major transcription start site) contained elements responsive to TGF-beta1 stimulation. The introduction of mutations into the putative consensus binding sequence for the Ets-family of transcription factors located at -133 greatly decreased the promoter activity responding to TGF-beta1 stimulation. The nuclear proteins extracted from TGF-beta1-stimulated HepG2 cells yielded a larger amount of DNA-nuclear protein complexes than did those extracted from unstimulated cells, as determined by an electrophoretic mobility shift assay using an oligonucleotide containing the Ets-site as a probe. These results suggest that TGF-beta1 stimulates HepG2 cells to express a higher level of alpha3 integrin by transcriptional upregulation via Ets transcription factors and to exhibit a more invasive phenotype.

Effects of combined therapy with interleukin 2 and interleukin 12 gene-transfected tumor vaccine for head and neck carcinoma.

BACKGROUND: The biological effects of cytokines are coming to be understood. The therapeutic effects of interleukin (IL) 2, IL-12, and interferon gamma (IFN-gamma) in cancer treatment have been reported, but there are problems when these cytokines are systemically used as therapeutic agents. OBJECTIVE: To examine the efficacy of IL-2 and IL-12 gene-transfected tumor cell vaccines for head and neck squamous cell carcinoma (SCC). METHODS: Homozygous mice with the autosomal recessive nude gene (BALB/c nu/nu mice) were inoculated subcutaneously in the right flank with cells from a human oral floor SCC cell line (KB cells). The mice were then injected with IL-2 and IL-12 gene-transfected KB cells (KB/IL-2 and KB/IL-12 cells, respectively) irradiated with 2000 rad (20 Gy). RESULTS: No mice died soon after the injection of the gene immunotherapy. The treatment with either KB/human IL-2 (hIL-2) or KB/murine IL-12 (mIL-12) was not very effective. However, the treatment with both KB/hIL-2 and KB/mIL-12 cells significantly and safely inhibited the growth of established tumors (P =.04). There was no significant difference in antitumor effect between once-weekly and twice weekly injections of both KB/hIL-2 and KB/mIL-12 cells. CONCLUSION: Double gene immunotherapy is safe and effective treatment for SCC in mice.