Efficient expression system of human recombinant laminin-5

Laminin-5, a heterotrimer of laminin alpha3, beta3, and gamma2 chains, is an essential component of various epithelial basement membranes, and it strongly promotes cellular adhesion and motility in vitro. In this study, we established an efficient expression system of human recombinant laminin-5 (rLN5), in which full-length cDNAs encoding the human laminin alpha3, beta3, and gamma2 chains were introduced into the human embryonic kidney cell line HEK293. rLN5 was purified from the conditioned medium of the HEK293 transfectant (LN5-HEK) by immuno-affinity chromatography in a yield of 1 mg protein/liter, about 10 times higher than that of a natural LN5 from human gastric cancer cells. rLN5 was indistinguishable from the natural LN5 in its protein composition and biological activity. In addition, analysis of HEK293 transfectants expressing two exogenous LN5 subunits showed that the alpha3/gamma2 chains and the beta3/gamma2 chains, but not the alpha3/beta3 chains, were secreted as heterodimers, suggesting an important role of the gamma2 chain in the association of the three LN5 subunits. The expression system of rLN5 can be used as an important tool to understand the biological functions of this laminin and may be applicable to future regenerative medicine.     

Assessing the Role of Cell-Surface Molecules in Central Synaptogenesis in the Drosophila Visual System

A hallmark of the central nervous system is its spatial and functional organization in synaptic layers. During neuronal development, axons form transient contacts with potential post-synaptic elements and establish synapses with appropriate partners at specific layers. These processes are regulated by synaptic cell-adhesion molecules. In the Drosophila visual system, R7 and R8 photoreceptor subtypes target distinct layers and form en passant pre-synaptic terminals at stereotypic loci of the axonal shaft. A leucine-rich repeat transmembrane protein, Capricious (Caps), is known to be selectively expressed in R8 axons and their recipient layer, which led to the attractive hypothesis that Caps mediates R8 synaptic specificity by homophilic adhesion. Contradicting this assumption, our results indicate that Caps does not have a prominent role in synaptic-layer targeting and synapse formation in Drosophila photoreceptors, and that the specific recognition of the R8 target layer does not involve Caps homophilic axon-target interactions. We generated flies that express a tagged synaptic marker to evaluate the presence and localization of synapses in R7 and R8 photoreceptors. These genetic tools were used to assess how the synaptic profile is affected when axons are forced to target abnormal layers by expressing axon guidance molecules. When R7 axons were mistargeted to the R8-recipient layer, R7s either maintained an R7-like synaptic profile or acquired a similar profile to r8s depending on the overexpressed protein. When R7 axons were redirected to a more superficial medulla layer, the number of presynaptic terminals was reduced. These results indicate that cell-surface molecules are able to dictate synapse loci by changing the axon terminal identity in a partially cell-autonomous manner, but that presynapse formation at specific sites also requires complex interactions between pre- and post-synaptic elements.     

Effect of auxins on the formation of ubiquinone by tobacco plant cells in suspension culture

Ubiquinone (UQ) formation in BY-2 tobacco cells was especially promoted by a high concentration of 2,4-D. 2,4,5-T, MCP and NAA also promoted UQ formation in these cells. The UQ content in the cells cultured at high concentrations of 2,4-D was higher than that of controls throughout the culture period. The addition of 2,4-D at an early period in cell growth was very effective in promoting UQ formation, but addition at the stationary phase was ineffective. Cell growth was improved by adding phosphate to the medium but UQ content was decreased. UQ content decreased slowly during subculturing, whereas cell growth recovered gradually.     

An Evidence for the Repair of Double-Strand Scissions in DNA of Micrococcus radiodurans after Alpha Bombardment

The conversion of prochaetoglobosins as plausible precursors into mycotoxin chaetoglobosin A (1) in a cell-free system of Chaetomium subaffine was unsuccessful. However, reductase activity of the 20-keto-analogues (1), and prochaetoglobosins II (5) and III (6) were found in a microsomal fraction of this fungi. Two new metabolites of chaetoglobosins, named chaetoglobosin F_ex (2) and 20-dihydro-chaetoglobosin A (3), were also isolated from the same micro-organisms. Their structures were elucidated by spectroscopic data and chemical transformation.     

Unusual distribution of mitochondrial large subunit rRNA in the cytosol during conjugation in Tetrahymena thermophila

The distribution of mitochondria during conjugation of the ciliated protozoan Tetrahymena thermophila was surveyed using a mitochondrial stain and fluorescence in situ hybridization (FISH). When the mitochondria-specific stain, Mito-Tracker, was used, the majority of mitochondria were detected in the cortex; their distribution was not changed during conjugation. On the other hand, FISH using mitochondrial large subunit (LSU) rRNA as a probe showed an unusual distribution of signals during conjugation. Unexpectedly, the signals were detected throughout the cytoplasm of conjugating cells. These signals were not observed in pre-mating cells and in exconjugants. The cytosolic localization of mitochondrial rRNA was supported by northern blot analysis using post-mitochondrial RNA fraction at the later stages of conjugation. These observations suggest selective mitochondrial breakdown or transport of LSU rRNA into cytosol. The biological significance of the conjugation-specific appearance of the cytosolic mitochondrial rRNA is discussed.     

5-hydroxytryptamine synthesis in HL-1 cells and neonatal rat cardiocytes

Some reports showed that serotonergic system might have existed and that 5-hydroxytryptamine (5-HT) was detected in the hamster heart. The source of 5-HT in the heart, however, remains to be fully elucidated. So the present study was designed to define serotonergic system and to clarify which cell could produce 5-HT in the heart. As a result, 5-HT was detected in homogenates of HL-1 cardiomyocytes by high performance liquid chromatography with fluorescence detection, but not in those of neonatal rat non-cardiomyocytes (NMCs). And TPH and AADC mRNAs were expressed in HL-1 cardiomyocytes and neonatal rat cardiomyocytes (MCs), not in NMCs. mRNAs of 5-HT(2A) receptor were detected in both MCs and NMCs, and those of 5-HT(2B) receptor in NMCs. These findings definitively demonstrate that 5-HT is secreted from the myocytes of the heart and strongly implied that 5-HT might play a certain role in cardiac physiology.     

Impact of Histone H1 on the Progression of Allergic Rhinitis and Its Suppression by Neutralizing Antibody in Mice

Nuclear antigens are known to trigger off innate and adaptive immune responses. Recent studies have found that the complex of nucleic acids and core histones that are derived from damaged cells may regulate allergic responses. However, no fundamental study has been performed concerning the role of linker histone H1 in mast cell-mediated type I hyperreactivity. In this study, we explored the impact of histone H1 on mast cell-mediated allergic responses both in vitro and in vivo. In the course of a bona-fide experimental allergen sensitization model upon co-injection with alum adjuvant, ovalbumin (OVA), but not PBS, induced elevated levels of circulating histone H1. Intranasal challenge with histone H1 to OVA/alum- (but not PBS/alum)-sensitized mice induced significantly severer symptoms of allergic rhinitis than those in mice sensitized and challenged with OVA. A monoclonal antibody against histone H1 not only suppressed mast cell degranulation, but also ameliorated OVA-induced nasal hyperreactivity and IgE-mediated passive cutaneous anaphylaxis. Our present data suggest that nuclear histone H1 represents an alarmin-like endogenous mediator acting on mast cells, and that its blockage has a therapeutic potential for mast cell-mediated type I hyperreactivity.     

Asialoglycoprotein receptor deficiency in mice lacking the major receptor subunit. Its obligate requirement for the stable expression of oligomeric receptor

The asialoglycoprotein receptor is an abundant hetero-oligomeric endocytic receptor that is predominantly expressed on the sinusoidal surface of the hepatocytes. A number of physiological and pathophysiological functions have been ascribed to this hepatic lectin (HL), the removal of desialylated serum glycoproteins and apoptotic cells, clearance of lipoproteins, and the sites of entry for hepatotropic viruses. The assembly of two homologous subunits, HL-1 and HL-2, is required to form functional, high affinity receptors on the cell surface. However, the importance of the individual subunits for receptor transport to the cell surface is controversial. We have previously generated HL-2-deficient mice and showed that the expression of HL-1 was significantly reduced, and the functional activity as the asialoglycoprotein receptor was virtually eliminated. However, we failed to detect phenotypic abnormalities. To explore the significance of the major HL-1 subunit for receptor expression and function in vivo, we have disrupted the HL-1 gene in mice. Homozygous HL-1-deficient animals are superficially normal. HL-2 expression in the liver is virtually abrogated, indicating that HL-1 is strictly required for the stable expression of HL-2. Although these mice are almost unable to clear asialo-orosomucoid, a high affinity ligand for asialoglycoprotein receptor, they do not accumulate desialylated glycoproteins or lipoproteins in the plasma.     

Flesh‐eating Streptococcus pyogenes triggers the expression of receptor activator of nuclear factor‐κB ligand

Human CD46 is a receptor for the M protein of group A streptococcus (GAS). The emm1 GAS strain GAS472 was isolated from a patient suffering from streptococcal toxic shock‐like syndrome. Human CD46‐expressing transgenic (Tg) mice developed necrotizing fasciitis associated with osteoclast‐mediated progressive and severe bone destruction in the hind paws 3 days after subcutaneous infection with 5 × 10⁵ colony‐forming units of GAS472. GAS472 infection induced expression of the receptor activator of nuclear factor‐κB ligand (RANKL) while concomitantly reducing osteoprotegerin expression in the hind limb bones of CD46 Tg mice. Micro‐computed tomography analysis of the bones suggested that GAS472 infection induced local bone erosion and systemic bone loss in CD46 Tg mice. Because treatment with monoclonal antibodies (mAbs) against mouse CD4⁺ and CD8⁺ T lymphocytes did not inhibit osteoclastogenesis, T lymphocyte‐derived RANKL was not considered a major contributor to massive bone loss during GAS472 infection. However, immunohistochemical analysis of the hind limb bones showed that GAS472 infection stimulated RANKL production in various bone marrow cells, including fibroblast‐like cells. Treatment with a mAb against mouse RANKL significantly inhibited osteoclast formation and bone resorption. These data suggest that increased expression of RANKL in heterogeneous bone marrow cells provoked bone destruction during GAS infection.