Simultaneous loss of phospholipase Cδ1 and phospholipase Cδ3 causes cardiomyocyte apoptosis and cardiomyopathy.

Phospholipase C (PLC) is a key enzyme in phosphoinositide turnover. Among 13 PLC isozymes, PLCδ1 and PLCδ3 share high sequence homology and similar tissue distribution, and are expected to have functional redundancy in many tissues. We previously reported that the simultaneous loss of PLCδ1 and PLCδ3 caused embryonic lethality because of excessive apoptosis and impaired vascularization of the placenta. Prenatal death of PLCδ1/PLCδ3 double-knockout mice hampered our investigation of the roles of these genes in adult animals. Here, we generated PLCδ1/PLCδ3 double-knockout mice that expressed PLCδ1 in extra-embryonic tissues (cDKO mice) to escape embryonic lethality. The cDKO mice were born at the expected Mendelian ratio, which indicated that the simultaneous loss of PLCδ1 and PLCδ3 in the embryo proper did not impair embryonic development. However, half of the cDKO mice died prematurely. In addition, the surviving cDKO mice spontaneously showed cardiac abnormalities, such as increased heart weight/tibial length ratios, impaired cardiac function, cardiac fibrosis, dilation, and hypertrophy. Predating these abnormalities, excessive apoptosis of their cardiomyocytes was observed. In addition, siRNA-mediated simultaneous silencing of PLCδ1 and PLCδ3 increased apoptosis in differentiated-H9c2 cardiomyoblasts. Activation of Akt and protein kinase C (PKC) θ was impaired in the hearts of the cDKO mice. siRNA-mediated simultaneous silencing of PLCδ1 and PLCδ3 also decreased activated Akt and PKCθ in differentiated-H9c2 cardiomyoblasts. These results indicate that PLCδ1 and PLCδ3 are required for cardiomyocyte survival and normal cardiac function.

Dendritic morphogenesis of cerebellar Purkinje cells through extension and retraction revealed by long-term tracking of living cells in vitro.

Cerebellar Purkinje cells have the most elaborate dendritic trees among the neurons in the CNS. To investigate the dynamic aspects of dendritic morphogenesis of Purkinje cells, we performed a long-term analysis of living cells in cerebellar cell cultures derived from glutamate decarboxylase 67-green fluorescent protein mice. Most Purkinje cells had several primary dendrites during the 25-day culture period. Repeated observation of green fluorescent protein-expressing Purkinje cells over a period of 10-25 days in vitro demonstrated that not only extension, but also retraction of primary dendrites occurred during this culture period. Interestingly, both extension and retraction of primary dendrites were active between 10 and 15 days in vitro, and retraction of a primary dendrite occurred concomitantly with elongation of other primary dendrites in the same cell. Analysis of the morphological characteristics of the retracted primary dendrites demonstrated that shorter and less branched primary dendrites tended to retract. Furthermore, treatment with an inhibitor of calcium/calmodulin-dependent protein kinase II reduced the number of primary dendrites specifically during 5-15 days in vitro, the culture period when the extension and retraction of primary dendrites occurred actively. Blockade of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate-type glutamate receptors also reduced the number of primary dendrites during the same culture period, while inhibition of glutamate transporters increased the number. These findings suggest that the final morphology of Purkinje cells is achieved not only through extension, but also through retraction of their dendrites, and that calcium/calmodulin-dependent protein kinase II and neuronal activity are involved in this dendritic morphogenesis.

Total synthesis and antifungal activity of 9-methoxystrobilurin L as the originally proposed 1,4-benzodioxan structure.

Total synthesis of both enantiomers of 9-methoxystrobilurin L as the originally proposed 1,4-benzodioxan structure was successfully achieved. The 1H and 13C NMR spectra of synthesized 9-methoxystrobilurin L were compared with those of a naturally-occurring sample. It was strongly indicated that naturally-occurring 9-methoxystrobilurin L has not the originally reported 1,4-benzodioxan structure but a 1,5-benzodioxepin structure, the same as previously reported 9-methoxystrobilurin K. Antifungal activities of the synthesized compounds toward several typical fungi were also examined, and they were less active than 9-methoxystrobilurin K.

In situ detection of activated caspase-3 in apoptotic granule neurons in the developing cerebellum in slice cultures and in vivo.

Some granule neurons naturally undergo apoptosis in the external granular layer (EGL) of the postnatally developing cerebellum. In the present study, we examined the involvement of caspase-3 in this apoptosis using an organotypic slice culture system of postnatal rat cerebellum and an antibody specific for the active form of caspase-3 (p20/17). Double staining by immunohistochemistry against p20/17 and in situ nick-end labeling showed that p20/17 was present in some of the apoptotic EGL neurons. A similar staining pattern was also observed in the postnatal cerebellum in vivo. Double positive cells were observed more frequently when T7 DNA polymerase was used for the DNA fragmentation labeling in place of terminal deoxynucleotidyl transferase, by which apoptotic cells at earlier stages were thought to be labeled. Taken together, whereas caspase-3 was shown to be activated in some of the apoptotic EGL neurons in the developing cerebellum, activation of caspase-3 in some apoptotic EGL neurons may occur before they become positive on DNA fragmentation labeling. In addition, there may be another mechanism of EGL neuron apoptosis that is independent of caspase-3.

Notch2 expression negatively correlates with glial differentiation in the postnatal mouse brain.

Notch family molecules are thought to be negative regulators of neuronal differentiation in early brain development. After expression in the embryonic period, Notch2 continues to be expressed postnatally in the specific regions in the rodent brain. Here, we examined Notch2 expression in the postnatal mouse brain using lacZ knockin animals at the Notch2 locus. Notch2 expression was observed in the developing cerebellum and hippocampus, characteristic regions where neurogenesis persists after birth. Double staining of sections revealed that Notch2 was expressed by Bergmann glia in the cerebellum, radial glia in the hippocampus, and some astrocytes in both regions. Notch2 expression by glial cells was clearly confirmed in dissociated cell cultures. Interestingly, neocortical glia, many of which did not express Notch2 in vivo, did express Notch2 in a dissociated culture condition. The triple staining of dissociated cell cultures revealed that stronger Notch2 expression correlated with the immature type of glial gene expressions: stronger vimentin and weaker glial fibrillary acidic protein expressions. In addition, Notch2 expression correlated with the incorporation of bromodeoxyuridine both in vivo and in vitro. Thus, these findings demonstrate that Notch2 is expressed not only by neuronal cells in the embryonic brain, but also by glial cells in the postnatal brain, and that its expression negatively correlates with glial differentiation, proposing its novel function as a negative regulator of glial differentiation in mammalian brain development.

Interleukin-10 inhibits both production of cytokines and expression of cytokine receptors in microglia.

Microglia, macrophage-like cells in the CNS, are multifunctional cells; they play an important role in removal of dead cells or their remnants by phagocytosis in the CNS degeneration and are one of important cells in the CNS cytokine network to produce and respond to a variety of cytokines. The functions of microglia are regulated by inhibitory cytokines. We have reported the expression of interleukin (IL)-10, one of the inhibitory cytokines, and its receptor in mouse microglia; therefore, IL-10 may affect microglial functions. In this study, we investigated the effects of IL-10 on purified microglia in culture. IL-10 inhibited lipopolysaccharide-induced IL-1beta and tumor necrosis factor-alpha production, lysosomal enzyme activity, and superoxide anion production in a dose-dependent manner, but did not affect granulocyte/ macrophage colony-stimulating factor-dependent proliferation of microglia. IL-10 also decreased the expression of both IL-6 receptor and lipopolysaccharide-induced IL-2 receptor but not IL-4 receptor on microglia as measured by flow cytometric analysis with an indirect immunofluorescence technique. IL-10 also decreased mRNA expression of IL-2 and IL-6 cytokine receptors. These results suggest that IL-10 is a unique and potent inhibitory factor in the CNS cytokine network involved in decreasing the expression of cytokine receptors as well as cytokine production by microglia.

Evidence for a critical role of DNA topoisomerase IIalpha in drug sensitivity revealed by inducible antisense RNA in a human leukaemia cell line.

To examine the role of human DNA topoisomerase IIalpha (topo IIalpha) in drug resistance, we selectively inhibited topo IIalpha gene expression in U937 human monocytic leukaemia cells stably transfected with a plasmid that allowed for Zn-mediated conditional expression of a human alpha-topo IIalpha antisense sequence. Expression of topo IIalpha mRNA was reduced to <30%, whereas no significant alteration of topo IIbeta mRNA expression was observed. Under these conditions, drug sensitivity to the topo-II-directed agents, etoposide and daunorubicin, was reduced to approximately 50%, whereas sensitivity to 4-hydroperoxy-cyclophosphamide (4-HC) was not altered. This suggests that a reduced amount of topo IIalpha mRNA may be sufficient for the resistance to topo II inhibitors in leukaemia cells.

Insulin-like growth factor-I analogue prevents apoptosis mediated through an interleukin-1 beta converting enzyme (caspase-1)-like protease of cerebellar external granular layer neurons: developmental stage-specific mechanisms of neuronal cell death.

Using an organotypic slice culture system of neonatal rat cerebellum, we examined developmental stage-specific mechanisms of cell death of granule neurons. This culture system allows a serial process of granule neuron development including their proliferation during the early culture period and the proceeding migration from the external granular layer to the internal granular layer in the presence of a supraphysiological concentration (5 micrograms/ml) of insulin. Insulin deprivation induced apoptosis of granule neurons in external granular layer but not in internal granular layer. A truncated analogue of insulin-like growth factor-I (des (1-3) insulin-like growth factor-I) prevented this apoptosis at a concentration of 65-650 ng/ml. Some apoptotic granule neurons expressed proliferating cell nuclear antigen but not TAG-1, a marker protein of the postmitotic and premigratory granule neurons. Thus, this apoptosis occurred at a specific stage in granule neuron development: at the stage before TAG-1 expression and at least partly at the proliferative state. Ac-YVAD-CHO, an inhibitor of interleukin-1 beta converting enzyme (caspase-1)-like proteases, had a protective effect on this apoptosis. Interleukin-1 beta converting enzyme (caspase-1)-like protease activity increased under the apoptosis-induced condition. High concentration of K+, which is known to prevent granule neuron apoptosis in dissociated cultures, had a partial protective effect on this apoptosis. These findings suggest that (i) cerebellar granule neurons fall into apoptosis at the specific developmental stage unless stimulated by insulin-like growth factor-I (analogue), (ii) this apoptosis is mediated through an interleukin-1 beta converting enzyme-like protease, and (iii) this apoptosis consists of K(+)-sensitive and K(+)-insensitive components.

Immunohistochemical analysis of developmental stage of external granular layer neurons which undergo apoptosis in postnatal rat cerebellum.

Some granule neurons naturally undergo apoptosis in the external granular layer (EGL) of the postnatally developing rat cerebellum. We analyzed the developmental stage-specificity of this apoptosis using double staining by in situ nick end labeling and immunohistochemistry against three proteins expressed at specific stages of granule neuron development. The amount of apoptosis of EGL neurons peaked on postnatal day 9. In the 9-day-old rat cerebellum, 54.0% of apoptotic EGL neurons expressed proliferating cell nuclear antigen. On the other hand, 22.2 and 15.4% of apoptotic EGL neurons existed in the postmitotic and premigratory zone defined by expression of TAG-1 and 440 kDa ankyrinB, respectively. Thus, proliferative granule neurons undergo apoptosis more frequently than postmitotic granule neurons in EGL of the developing cerebellum. This suggests that there are developmental stage-specific mechanisms of apoptosis of cerebellar granule neurons.

Migration activity of microglia and macrophages into rat brain.

We examined the entry of intra-arterially injected microglia and macrophages into the brain using a rat muscle graft model to compare their respective abilities to invade the brain parenchyma. Isolated microglia without any activation treatment entered into the brain with or without the muscle graft, while macrophages activated by phorbol 12-myristate-13-acetate (PMA) entered the brain only in the presence of the muscle graft. These results suggest that microglia have a higher affinity for the brain than macrophages.

Fusion of the platelet-derived growth factor receptor beta to a novel gene CEV14 in acute myelogenous leukemia after clonal evolution.

Chromosomal translocations involving band 5q31-35 occur in several hematologic disorders. A clone with a t(5; 14)(q33; q32) translocation appeared at the relapse phase in a patient with acute myelogenous leukemia who exhibited a sole chromosomal translocation, t(7; 11), at initial diagnosis. After the appearance of this clone, the leukemia progressed with marked eosinophilia, and combination chemotherapy was ineffective. Southern blot analysis showed a rearrangement of the platelet-derived growth factor receptor beta (PDGFRbeta) gene at 5q33 which was not observed at initial diagnosis. This translocation resulted in a chimeric transcript fusing the PDGFRbeta gene on 5q33 with a novel gene, CEV14, located at 14q32. Expression of the 5' region of the PDGFRbeta cDNA, upstream of the breakpoint, was not detected. However, the 3' region of PDGFRbeta, which was transcribed as part of the CEV14-PDGFRbeta fusion gene, was detected. A partial cDNA for a novel gene, CEV14, includes a leucine zipper motif and putative thyroid hormone receptor interacting domain and is expressed in a wide range of tissues. The expression of a CEV14-PDGFRbeta fusion gene in association with aggressive leukemia progression suggests that this protein has oncogenic potential.

Detection of polymorphic FGF receptor 1 mRNA in bovine lens epithelium by PCR.

The expression of FGFR 1, 2 and 3 but not FGFR 4 was detected in bovine lens epithelial cells. FGFR 1 expression of both the 2-lg and 3-lg forms was detected by a difference in the lengths of the PCR products. Furthermore, mRNA which included or excluded six nucleotides corresponding to two amino acids in the acid box region of FGFR 1 was detected by a difference in mobility of the homo- and hetero-duplex strands formed in the PCR reaction.

Transient expression of M-CSF is important for osteoclast-like cell differentiation in a monocytic leukemia cell line.

Cells of U937, a human monocytic leukemia cell line, differentiate into macrophages by treatment with 12-o-tetradecanoylphorbol-13-acetate (TPA), whereas cells treated with 1 alpha, 25-dihydroxyvitamin D3 [1,25-(OH)2D3] continue to grow without undergoing differentiation. When U937 cells were successively treated with TPA and 1,25-(OH)2D3, tartrate-resistant acid phosphatase-positive multinucleated cells appeared at 5 days after the treatment. These osteoclast-like cells released a soluble form of 45Ca from 45Ca-labeled bone particles. These cells were not formed when the order of treatment with TPA and 1,25-(OH)2D3 was reversed. Use of either dexamethasone or interferon-gamma (IFN-gamma) was effective in inhibiting the formation of these osteoclast-like cells. The expression of c-src, c-fms, and macrophage colony stimulating factor (M-CSF) was induced by TPA treatment; however, TPA-induced M-CSF gene transcription was attenuated by the subsequent addition of 1,25-(OH)2D3. Furthermore, both dexamethasone and IFN-gamma impaired the attenuation of M-CSF expression, suggesting that the transient expression of M-CSF may be important for the formation of osteoclast-like cells.

Expression of Ly-6C on microglia in the developing and adult mouse brain.

Expression of Ly-6C, a murine homolog of human CD59, in the brain was examined immunologically using an ER-MP20 monoclonal antibody both in vitro and in vivo. Ly-6C was expressed both on the isolated microglia and on microglia in the developing and adult cerebellum. The number of cerebellar microglia expressing Ly-6C markedly increased during the first postnatal week in mouse development. The expression of Ly-6C on microglia in the developing and adult brain may be related to the unique role(s) of microglia under normal and/or pathological conditions of the brain.

Transcriptional and post-transcriptional regulation of pr22 (Op18) with proliferation control.

The pr22 gene was isolated as a gene which is expressed in proliferating cells but not in cells which are differentiated or growth-arrested. When cells of the human monocytic cell line, U937, were differentiated into macrophages, transcription of pr22 was almost completely suppressed. Serum starvation resulted in the inhibition of transcription, although U937 failed to differentiate. In a culture synchronized with excess thymidine, mRNA of pr22 was detected at the G1/S boundary, with the level increasing in the S phase and decreasing in the G2 phase. The gene product, pr22 protein (Pr22) was found to be identical to Op18 as well as to a catastrophe factor. Genes homologous to pr22 were detected in the genome of mouse but not in that of yeast, or Drosophila. The 5' up-stream region of the genomic pr22 contained CpG islands but no TATA box at its appropriate position. About 20% of cell nuclei of normal human fibroblasts were stained in a speckled manner with a monoclonal antibody for C-terminal peptide of Pr22, and these cells were found to be in phases S and G2. The mitotic apparatus was also strongly stained. By Western blot analysis, Pr22 was detected in the nuclear fraction but not in the cytoplasm. The level increased from middle S to G2 phase and remained high until the early G1 phase. N-terminal truncated Pr22 was also detected in these phases. These results suggest that Pr22 may have an additional role other than just functioning in association with microtubules.

Selective induction of interleukin-6 in mouse microglia by granulocyte-macrophage colony-stimulating factor.

Astrocytes produce granulocyte/macrophage colony-stimulating factor (GM-CSF) and support the survival and proliferation of microglia. To study the functions of GM-CSF in the central nervous system (CNS), we examined the effects of GM-CSF on cytokine production by glial cells. GM-CSF induced interleukin-6 (IL-6) production by microglia, but not by astrocytes, in a dose-dependent manner as assessed by bioassay and the detection of IL-6 mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. GM-CSF did not induce tumor necrosis factor (TNF) alpha or IL-1 in microglia and astrocytes, whereas lipopolysaccharide induced all these cytokines. The induction of IL-6 by GM-CSF in microglia was completely inhibited by antibodies to GM-CSF. Neither IL-3 nor macrophage-CSF (M-CSF) induced IL-6 production in microglia. Given that IL-1 and TNF alpha, monokines derived from microglia, induce IL-6 production in astrocytes, but not in microglia, results indicate that astrocytes and microglia may mutually regulate IL-6 production by different cytokines.

Macrophage colony-stimulating factor (M-CSF) inhibits the decrease in the amount of rRNA and IA beta mRNA in cultured epidermal Langerhans cells of the mouse.

Macrophage colony-stimulating factor (M-CSF) is a keratinocyte-derived cytokine whose function in skin is not completely clarified. We investigated its effects on Langerhans cells by examining the amount of IA beta mRNA, beta-actin mRNA and rRNA per cell, and compared them with the effects of other cytokines such as granulocyte/macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha). After culture for 24 h in the absence of exogenous cytokines, rRNA in Langerhans cells decreased steeply while beta-actin mRNA increased. IA beta mRNA also decreased sharply. These decreases in the amount of rRNA and IA beta mRNA were limited when cytokines were added to the culture medium (in order of efficiency M-CSF > GM-CSF > TNF-alpha), but M-CSF was less potent than GM-CSF in up-regulating beta-actin mRNA (GM-CSF > M-CSF, TNF-alpha). The effect of M-CSF, but not that of GM-CSF, was restricted by simultaneous treatment of cells with TNF-alpha. None of these effects engendered a change in the viability of the Langerhans cells in a 24-hr culture. Reverse-transcribed polymerase chain reaction analysis demonstrated that c-fms, the gene of the M-CSF receptor, was expressed in Langerhans cells, implying the physiological importance of M-CSF in vivo. A protein kinase C activator, TPA, up-regulated the amount of IA beta mRNA, while a protein kinase C inhibitor, H-7, suppressed the effects of all three cytokines. These results suggest that M-CSF, in conjugation with TNF-alpha and GM-CSF, plays an important role in controlling the physiological state of Langerhans cells, probably through the activation of protein kinase C.

Telomere elongation observed in immortalized human fibroblasts by treatment with 60Co gamma rays or 4-nitroquinoline 1-oxide.

Telomeres are the tandemly repeated (TTAGGG)n sequences that make up the structural and functional ends of all chromosomes in mammals. Many lines of evidence indicate that telomeres stabilize chromosomes, prevent aberrant recombination, and direct chromosome attachment to the nuclear membrane. Since DNA polymerase requires a labile primer to initiate unidirectional 5'-3' DNA synthesis, some bases at the 3' end of each template strand are not copied unless special mechanisms bypass this end-replication problem. To overcome this problem, most eukaryotic cells use telomerase, an enzyme that elongates telomeres. However, this enzyme has not been detected in normal human cells, and these cells lose telomeres with cell division. Cellular senescence might be the result of this loss. Thus, activation of telomerase seems to be critical for the immortalization of human cell lines. In addition, substantial evidence indicates that immortalization in itself is a rate-limiting step for the malignant transformation of human cells. We have treated normal human fibroblasts (AD387, KMS-6, and OUMS-24 lines) intermittently with either 60Co gamma rays or 4-nitroquinoline 1-oxide (4NQO) during serial subcultivations, and have obtained three immortalized cell lines, SUSM-1, KMST-6, and OUMS-24F. In KMS-6 and OUMS-24, the mean terminal restriction fragment length significantly decreased as the population-doubling level increased. The rate of telomere loss was 40 and 50 bp/population doubling in the KMS-6 and OUMS-24 cell lines, respectively. Once these normal cell lines were immortalized, their telomeres became elongated. Similar data were obtained for AD387 cells and their immortalized SUSM-1 cells. These results suggest that telomeres play a critical role in cellular senescence and in the immortalization processes of human cells.

Expression pattern of messenger RNAs for prostanoid receptors in glial cell cultures.

Expression level of messenger RNAs (mRNAs) for prostanoid EP3, FP, and TP receptors was investigated in cultured rat astrocytes, oligodendrocytes, and microglia, as well as in meningeal fibroblasts, rat glioma C6 cells, rat pheochromocytoma PC12 cells, whole brain, and several peripheral tissues by reverse transcriptase-polymerase chain reaction. Cultured astrocytes and oligodendrocytes expressed mRNAs for 3 prostanoid receptors examined. In contrast, cultured microglia and pheochromocytoma PC12 cells expressed EP3 and TP receptor mRNAs, but not FP receptor mRNA. Glioma C6 cells expressed only TP receptor mRNA among 3 prostanoid receptors with the same expression level as that in astrocytes. Cultured meningeal fibroblasts expressed 3 receptor transcripts, and their expression levels were lower than those in astrocytes. Expression level of mRNA for each prostanoid receptor in cultured glial cells was higher than that in whole brain. These observations suggest that each prostanoid has its specific roles in each glial cell type of the brain.