Formation and characterization of spontaneously formed heterokaryons between quail myoblasts and 3T3-L1 preadipocytes: correlation between differential plasticity and degree of differentiation

Skeletal muscle cells and adipose cells have a close relationship in developmental lineage. Our previous study has shown that the heterokaryons between quail myoblasts and undifferentiated 3T3-L1 cells (preadipocytes) normally differentiated into myotubes, whereas the heterokaryons between myoblasts and differentiated 3T3-L1 cells (adipocytes) failed myogenic differentiation. These results suggest differences between preadipocytes and adipocytes. The purpose of this study was to clarify whether preadipocytes have flexibility in differentiation before terminal adipose differentiation. Presumptive quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells) and mouse 3T3-L1 cells (either preadipocytes or adipocytes) were co-cultured for 48 h under conditions allowing myogenic differentiation. On co-culture between myoblasts and undifferentiated 3T3-L1 cells, heterokaryotic myotubes formed spontaneously, but not on co-culture with differentiated 3T3-L1 cells. In addition, the heterokaryotic myotubes expressed mouse myogenin derived from the 3T3-L1 cell gene. Our previous study indicated that the fusion sensitivity of differentiating myoblasts change with decreasing cholesterol of the cell membrane during myoblast fusion. Thus we compared the level of membrane cholesterol between undifferentiated and differentiated 3T3-L1 cells. The result showed that the level of membrane cholesterol in 3T3-L1 cells increases during adipose differentiation. Corresponding to the increase in membrane cholesterol content, differentiated 3T3-L1 cells had lower sensitivity to HVJ (Sendai virus)-mediated cell fusion than undifferentiated 3T3-L1 cells. This study demonstrated that 3T3-L1 cells at an undifferentiated state have a capacity for spontaneous fusion with differentiating myoblasts following myogenic differentiation, and that the capacity is lost after terminal adipose differentiation.     

Amino Acid Requirements for the Growth of Aedes albopictus Clone C6/36 Cells and for the Production of Dengue and Chikungunya Viruses in the Infected Cells

Amino acid requirements for the growth of Aedes albopictus, clone C6/36, cells and for the production of dengue (DEN) and Chikungunya (CHIK) viruses were examined by growing the cells or the viruses in media which were deprived of one of the 20 amino acids. Cell growth was markedly inhibited when cystine was omitted from the medium, and to a lesser extent by arginine deprivation. On the other hand, omission of alanine, asparagine, aspartic acid, and glutamic acid at the same time did not affect cell growth. Marked accumulation of alanine was observed in the medium when the cells were grown for 8 days in complete medium, with concomitant depletion of aspartic acid and glutamic acid. The production of CHIK virus was inhibited markedly by omission of cystine from the medium after virus infection, while the production of DEN viruses was more affected by glycine deprivation, although cystine deprivation also inhibited virus production to a lesser extent. On the other hand, production of CHIK and DEN viruses was not affected when alanine, asparagine, aspartic acid, and glutamic acid were omitted from the medium at the same time.     

Abnormal production of macrophage inflammatory protein-1α by microglial cell lines derived from neonatal brains of Sandhoff disease model mice

Sandhoff disease (SD) is a lysosomal β-hexosaminidase deficiency involving excessive accumulation of undegraded substrates, including terminal N -acetylglucosamine-oligosaccharides and GM2 ganglioside, and progressive neurodegeneration. Our previous study demonstrated remarkable induction of macrophage inflammatory factor-1α (MIP-1α) in microglia in the brains of SD model mice as a putative pathogenic factor for SD via microglia-mediated neuroinflammation. In this study, we established microglial cell lines (WT- and SD-Mg) from wild-type and SD mice, and first demonstrated the enhanced production of MIP-1α in SD-Mg. Inhibitors of protein kinase C (PKC) and Akt reduced the production of MIP-1α by SD-Mg. Elevated activation of Akt and partial translocation of PKC isozymes (α, βI, βII, and δ) from the cytoplasm to the membrane in SD-Mg were also revealed by means of immunoblotting. Furthermore, it was demonstrated that intracellular extracellular signal-regulated kinase, c-Jun N-terminal kinase, and phospholipase C (PLC), but not phosphoinositide 3-kinase, should contribute to the induction of MIP-1α in SD-Mg, and that PLC could independently regulate the activation of both PKC and Akt. We proposed here that the deregulated activation of PLC should cause the enhanced MIP-1α production via plural signaling pathways mediated by PKC and Akt, followed by extracellular signal-regulated kinase and c-Jun N-terminal kinase, in SD-Mg.     

Alien grasses in Brazilian savannas: a threat to the biodiversity

African grasses used as forage are spreading fast in cerrado (Brazilian savanna) patches, probably displacing native species. An analysis of the graminoid species abundance was performed in Cerrado Pé-de-Gigante Reserve (São Paulo State, Brazil), where their relative frequency, density, dominance and the value of importance were assessed in two cerrado forms: cerrado sensu stricto (denser) and campo cerrado (open). Thirty-six transects were determined, along which 3240.5 m × 0.5 m herbaceous samples were taken. Ordination by CCA analysis was performed to detect gradients in the graminoid species distribution, according to shading, distance from the reserve border and aspect. Interspecific associations among the species were tested. A total of 93 species were sampled, predominantly Poaceae and Myrtaceae families. Two alien grasses were found, Melinis minutiflora and Brachiaria decumbens, with very high values of importance. Light availability proved to be the most important analyzed environmental factor related to graminoid distribution, strongly correlated with the abundance of M. minutiflora. Both alien grasses were negatively associated with most native graminoids, suggesting they exert a strong competitive pressure on the native herbaceous community. Attention must be taken to the introduction of alien species in the country.     

Mechanism of abnormal growth in astrocytes derived from a mouse model of GM2 gangliosidosis

P2X receptors mediate a variety of physiological actions, including smooth muscle contraction, neuro-endocrine secretion and synaptic transmission. Among P2X receptors, the P2X₃ subtype is expressed in sensory neurons of dorsal root- and trigeminal-ganglia, where it performs a well-recognized role in sensory and pain transmission. Recent evidence indicates that the strength of P2X₃-mediated responses is modulated in vivo by altering the number of receptors at the plasma membrane. In the present study, we investigate the trafficking properties of P2X₃ receptor in transfected HEK293 cells and in primary cultures of dorsal root ganglion neurons, finding that P2X₃ receptor undergoes rapid constitutive and cholesterol-dependent endocytosis. We also show that endocytosis is accompanied by preferential targeting of the receptor to late endosomes/lysosomes, with subsequent degradation. Furthermore, we observe that at steady state the receptor localizes predominantly in lamp1-positive intracellular structures, with a minor fraction present at the plasma membrane. Finally, the level of functional receptor expressed on the cell surface is rapidly up-regulated in response to agonist stimulation, which also augments receptor endocytosis. The findings presented in this work underscore a very dynamic trafficking behavior of P2X₃ receptor and disclose a possible mechanism for the rapid modulation of ATP-mediated responses potentially relevant during physiological and pathological conditions.     

An immunohistochemical study on hard tissue formation in a subcutaneously transplanted rat molar

While dental pulp undergoes calcification following tooth replantation or transplantation, we actually know little about these mechanisms. We therefore conducted histological and immunohistochemical evaluations of mineralized tissue that formed in the pulp of rat maxillary molar transplanted into abdominal subcutaneous tissue. One, 2, 3, and 4 weeks post-transplantation, the teeth were investigated immunohistochemically using antibodies to osteocalcin (OCN), osteopontin (OPN), bone sialoprotein (BSP), dentin sialoprotein (DSP), and tissue non-specific alkaline phosphatase (TNAP). In the 1st week after transplantation, cell-rich hard tissue was formed at the root apex. At 2 weeks, formations of hard tissue, with few cells in the root canals and bone-like tissue in the coronal pulp chamber, were noted. After 3 and 4 weeks, the amounts of these hard tissues were increased. The immunolocalization of OCN, OPN, and BSP was seen strongly in coronal and apical hard tissues, but weakly in the root hard tissue. Conversely, DSP localized in the root hard tissue, but not in other newly formed hard tissues. At 1 week, TNAP localized along the periphery of the apical hard tissue and the lower surfaces of root predentin. These results demonstrate that the newly formed hard tissues in the pulp cavity of subcutaneously transplanted molars could be classified into three types, suggesting that these might be formed by type-specific cells.     

Characterization of heterokaryons between skeletal myoblasts and preadipocytes: myogenic potential of 3T3-L1 preadipocytes

It has been shown previously that heterokaryons between myoblasts and non-myogenic cells disturb myogenic differentiation (Hirayama et al. (2001); Cell Struct. Funct. 26, 37-47), suggesting that some myogenesis inhibitory factors exist in non-myogenic cells. Skeletal myoblasts and adipose cells are derived from a common mesodermal stem cell, indicating that both cells have a closer relationship in the developmental lineage than the other somatic cells. To investigate the functional relationship between myoblasts and adipose cells, heterokaryons between quail myoblasts and 3T3-L1 cells, a mouse preadipocyte cell line, were prepared and examined for characteristics of myogenic differentiation. Myogenic differentiation was inhibited in the heterokaryons between quail myoblasts and well-differentiated (adipocytes) 3T3-L1 cells. On the contrary, normal myogenic differentiation proceeded in the heterokaryons between quail myoblasts and undifferentiated (preadipocytes) 3T3-L1 cells. Further investigation showed that the mouse myogenin gene from 3T3-L1 cells was transactivated in the heterokaryons between quail myoblasts and undifferentiated 3T3-L1 cells. The results demonstrated that undifferentiated 3T3-L1 cells have no myogenesis inhibitory factors but acquire these during terminal differentiation into adipocytes.     

A Rapid Increase in the Level of Binding Protein (BiP) is Accompanied by Synthesis and Degradation of Storage Proteins in Pumpkin Cotyledons

The binding protein (BiP) has been implicated in cotranslationalfolding of nascent polypeptides, and in the recognition anddisposal of aberrant polypeptides. To elucidate the involvementof BiP in the biosynthesis of vacuolar proteins, we have characterizedthe protein in pumpkin cotyledons during seed maturation andseedling growth. Isolated microsomes from maturing pumpkin cotyledonscontained a significant amount of BiP, protein-disulfide isomeraseand calreticulin. We have purified a 70-kDa protein; sequencesof the N-terminus and internal fragments of this protein exhibiteda high identity to the sequence of soybean BiP. Immunoblot analysiswith specific antibodies raised against the purified BiP showedthat the amount of BiP in a cotyledon increased markedly atthe middle stages and then decreased. The increase was accompaniedby the synthesis of storage proteins and the development ofthe endoplasmic reticulum in the cotyledons at the middle stageof seed maturation. Most of these storage proteins degradeddramatically between 2 and 5 days after seed germination, andthe degradation was also accompanied by a rapid increase inthe level of BiP. Subcellular fractionation of the 4-day-oldcotyledons showed a high accumulation of BiP in the endoplasmicreticulum. It is possible that BiP might be involved in thesynthesis of seed storage proteins during maturation and inthe synthesis of hydrolytic enzymes responsible for the degradationof the storage proteins during seed germination. (Received September 18, 1996; Accepted January 8, 1997)