The enhancing action of D-galactosamine on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophage cells

The effect of D-galactosamine (D-GalN) on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells was examined. D-GalN augmented the production of NO, but not tumor necrosis factor (TNF)-alpha in LPS-stimulated RAW 264.7 cells. Pretreatment of D-GalN augmented the NO production whereas its post-treatment did not. D-GalN augmented the NO production in RAW 264.7 cells stimulated with either TNF-alpha and interferon-gamma. The augmentation of LPS-induced NO production by D-GalN was due to enhanced expressions of an inducible type of NO synthase mRNA and proteins. Intracellular reactive oxygen species (ROS) were exclusively generated in RAW 264.7 cells stimulated with D-GalN and LPS. Scavenging of intracellular ROS abrogated the augmentation of NO production. It was therefore suggested that D-GalN might augment LPS-induced NO production through the generation of intracellular ROS.     

Enzymatic characterization of scytalone dehydratase Val75Met variant found in melanin biosynthesis dehydratase inhibitor (MBI-D) resistant strains of the rice blast fungus

Carpropamid ((1RS,3SR)-2,2-dichloro-N-[(R)-1-(4-chlorophenyl)ethyl]-1-ethyl-3-methylcyclopropanecarboxamide) is a potent chemical against the rice blast fungus, Pyricularia oryzae. In 2001, isolates of the fungus with reduced sensitivity to this fungicide appeared in Saga Prefecture of Japan and were regarded as a potential threat to rice protection by carpropamid. The cause of the resistance has been identified genetically as a point mutation resulting in the Val75Met change in scytalone dehydratase, the primary target of the fungicide. We constructed an overexpression system of the variant enzyme and characterized the kinetics in the catalysis and the inhibition by carpropamid isomers. The variant enzyme retained a significant level of enzymatic activity. Inhibition of the variant enzyme by carpropamid was more than 200-fold reduced in comparison with that of the wild-type. Based on the results, here we propose possible mechanisms of the carpropamid-resistance of the variant enzyme in retaining the normal enzymatic activity.     

The distinct stage-specific effects of 2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid on the activation of MAP kinase and Cdc2 kinase in Xenopus oocyte maturation

2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid (PACA), pharmacological inhibitor of phospholipase A(2) (PLA(2)), inhibits epinephrine-stimulated thromboxane production in human platelets. In this study, we investigated the effect of PACA on meiotic maturation individually in stages V and VI oocytes. PACA prevented the maturation in stage V but merely delayed the process in stage VI oocytes. This was associated with the strong inhibition of Mos synthesis at both stages. Besides, PACA-induced inhibition of MAPK activation was evident in stage V but not in stage VI oocytes. PACA also inhibited the activation of Cdc2 kinase (Cdc2) in stage V but merely delayed the process in stage VI oocytes. Furthermore, 5 microM and higher concentrations of PACA completely inhibited the activation of MAPK and Cdc2 only in stage V, not in stage VI, oocytes. Moreover, we propose PACA as a new tool for the study of Xenopus oocyte maturation, which can also play a unique role for the studies of the stage-specific activation of MAPK and Cdc2.     

Induced in vitro differentiation of pancreatic-like cells from human amnion-derived fibroblast-like cells

There is growing evidence that the human amnion contains various types of stem cells. As amniotic tissue is readily available, it has the potential to be an important source of material for regenerative medicine. In the present study, we evaluated the potential of human amnion-derived fibroblast-like (HADFIL) cells to differentiate into pancreatic islet cells. Two HADFIL cell populations, derived from two different neonates, were analyzed. The expression of pancreatic cell-specific genes was examined before and after in vitro induction of cellular differentiation. We found that Pdx-1 , Isl-1 , Pax-4 , and Pax-6 showed significantly increased expression following the induction of differentiation. In addition, immunostaining demonstrated that insulin, glucagon, and somatostatin were present in HADFIL cells following the induction of differentiation. These results indicate that HADFIL cell populations have the potential to differentiate into pancreatic islet cells. Although further studies are necessary to determine whether such in vitro -differentiated cells can function in vivo as pancreatic islet cells, these amniotic cell populations might be of value in therapeutic applications that require human pancreatic islet cells.     

Novel Ca2+/calmodulin-dependent protein kinase expressed in actively growing mycelia of the basidiomycetous mushroom Coprinus cinereus

We isolated cDNA clones for novel protein kinases by expression screening of a cDNA library from the basidiomycetous mushroom Coprinus cinereus. One of the isolated clones was found to encode a calmodulin (CaM)-binding protein consisting of 488 amino acid residues with a predicted molecular weight of 53,906, which we designated CoPK12. The amino acid sequence of the catalytic domain of CoPK12 showed 46% identity with those of rat Ca2+/CaM-dependent protein kinase (CaMK) I and CaMKIV. However, a striking difference between these kinases is that the critical Thr residue in the activating phosphorylation site of CaMKI/IV is replaced by a Glu residue at the identical position in CoPK12. As predicted from its primary sequence, CoPK12 was found to behave like an activated form of CaMKI phosphorylated by an upstream CaMK kinase, indicating that CoPK12 is a unique CaMK with different properties from those of the well-characterized CaMKI, II, and IV. CoPK12 was abundantly expressed in actively growing mycelia and phosphorylated various proteins, including endogenous substrates, in the presence of Ca2+/CaM. Treatment of mycelia of C. cinereus with KN-93, which was found to inhibit CoPK12, resulted in a significant reduction in growth rate of mycelia. These results suggest that CoPK12 is a new type of multifunctional CaMK expressed in C. cinereus, and that it may play an important role in the mycelial growth.     

Mycobacteria Exploit Host Hyaluronan for Efficient Extracellular Replication

In spite of the importance of hyaluronan in host protection against infectious organisms in the alveolar spaces, its role in mycobacterial infection is unknown. In a previous study, we found that mycobacteria interact with hyaluronan on lung epithelial cells. Here, we have analyzed the role of hyaluronan after mycobacterial infection was established and found that pathogenic mycobacteria can grow by utilizing hyaluronan as a carbon source. Both mouse and human possess 3 kinds of hyaluronan synthases (HAS), designated HAS1, HAS2, and HAS3. Utilizing individual HAS-transfected cells, we show that HAS1 and HAS3 but not HAS2 support growth of mycobacteria. We found that the major hyaluronan synthase expressed in the lung is HAS1, and that its expression was increased after infection with Mycobacterium tuberculosis. Histochemical analysis demonstrated that hyaluronan profoundly accumulated in the granulomatous legion of the lungs in M. tuberculosis-infected mice and rhesus monkeys that died from tuberculosis. We detected hyaluronidase activity in the lysate of mycobacteria and showed that it was critical for hyaluronan-dependent extracellular growth. Finally, we showed that L-Ascorbic acid 6-hexadecanoate, a hyaluronidase inhibitor, suppressed growth of mycobacteria in vivo. Taken together, our data show that pathogenic mycobacteria exploit an intrinsic host-protective molecule, hyaluronan, to grow in the respiratory tract and demonstrate the potential usefulness of hyaluronidase inhibitors against mycobacterial diseases.     

An Internal View of the Spherical Body of Treponema macrodentium as Revealed by Scanning Electron Microscopy

The osmium-dimethyl sulfoxide-osmium method for clear visualization of intracellular structure was used to observe the detailed inner structure of the spherical bodies produced in vitro by a human oral treponeme. Scanning electron microscopy of the cracked spherical body revealed no morphological differences between the outer and inner surfaces of the spherical body membrane, and that multiple folded or somewhat linear main bodies adhere closely to the inner surface. In addition, axial flagella partially free from the main bodies spread widely within the body to make a network, and a number of blebs ranging from approximately 1 μm to 0.2 μm in diameter were located near the terminal or subterminal areas of the main bodies. The origin of the blebs and the mechanism of spherical body formation are discussed.