High resistance to oxygen radicals and heat is caused by a galactoglycerolipid in Microbacterium sp. M874

Microbacterium sp. M874 produced a glyceroglycolipid, di-O-12-methyl-tetradecanoyl-3-O-beta-D-galactopyranosyl-sn-glycerol, at about the 50 microM level. Though the strain was highly resistant to tertiary-butyl hydroperoxide (tBHP) in a glycolipid-productive medium, the resistance was reduced in a nonproductive medium. Exogenous addition of the glycolipid to the nonproductive culture restored the resistance. This addition also increased the resistance to heat, ethanol, and 4-chloro-1-naphthol, in which oxygen radicals might participate. The parallel relationship found in strain M874 mutants between glycolipid productivity and resistance to tBHP or heat suggested that the resistance was mainly caused by the glycolipid. On addition of the glycolipid to a glycolipid-nonproductive culture, it was immediately incorporated into the cells and functioned as an anti-oxygen radical reagent. Thereafter, its intracellular level remained largely unchanged for at least 5 h, even in the presence of tBHP, and its activity was maintained. The glycolipid at 142 microM was sufficient to prevent the cytotoxicity induced by 88.9 mM tBHP. The glycolipid production was not induced by pretreatment with a low level of tBHP or a sublethal heat shock. In brief, the glycolipid might play an essential role in the prevention of damage by oxygen radicals in the glycolipid-producing bacterium.     

Substance P-induced cadherin expression and its signal transduction in a cloned human corneal epithelial cell line

Although the absence of Substance P (SP), a neurotransmitter in the trigeminal nerve, has been speculated as a cause for developing neurotrophic keratitis, its exact pathogenesis is still not clarified. In a previous report, we showed with electron microscopic examination that epithelial cell attachment was weakened in denervated corneas. In this study, SV40-transformed human corneal epithelial cells (HCE-Ts) were used to explore the molecular mechanisms responsible for mediating regulation of E-cadherin expression in response to Substance P receptor stimulation. Expression of the mRNAs for specific SP receptors, neurokinin (NK)-1R, NK-2R, and NK-3R, was demonstrated with RT-PCR. The cells were treated with various concentrations of SP in vitro, and the expression of an adhesion molecule E-cadherin was analyzed by immunofluorescence, immunoblotting, and enzyme-linked immunosorbent assay (ELISA) using an anti-E-cadherin antibody. E-cadherin expression was increased by SP in a dose-dependent manner both in the cytosolic fraction and in the cell membrane fraction. This increase in E-cadherin expression was completely inhibited by Calphostin C (PKC inhibitor) and KN-62 (CaMK inhibitor), but not by H-89 (PKA inhibitor), indicating that SP-induced E-cadherin expression involves the activation of protein kinase C (PKC) and calmodulin kinase (CaMK). SP did not affect cell proliferation at all. All these findings indicate that SP induced E-cadherin expression through PKC and CaMK activation and suggest that a lack of SP may account in part for the pathogenesis of neurotrophic keratitis.     

Isolation of Medicago truncatula mutants defective in calcium oxalate crystal formation

Plants accumulate crystals of calcium oxalate in a variety of shapes, sizes, amounts, and spatial locations. How and why many plants form crystals of calcium oxalate remain largely unknown. To gain insight into the regulatory mechanisms of crystal formation and function, we have initiated a mutant screen to identify the genetic determinants. Leaves from a chemically mutagenized Medicago truncatula population were visually screened for alterations in calcium oxalate crystal formation. Seven different classes of calcium oxalate defective mutants were identified that exhibited alterations in crystal nucleation, morphology, distribution and/or amount. Genetic analysis suggested that crystal formation is a complex process involving more than seven loci. Phenotypic analysis of a mutant that lacks crystals, cod 5, did not reveal any difference in plant growth and development compared with controls. This finding brings into question the hypothesized roles of calcium oxalate formation in supporting tissue structure and in regulating excess tissue calcium.     

Apoptosis in skin pigment cells of the medaka, Oryzias latipes (Teleostei), during long-term chromatic adaptation: the role of sympathetic innervation

Many teleost fish can adapt their body color to a background color by changing the morphology and density of their skin pigment cells. Melanophore density in fish skin decreases during long-term adaptation to a white background. Although cell death, especially apoptosis, is thought to be involved in these morphological changes, there are no data clearly supporting this mechanism. Using medaka fish, Oryzias latipes, we observed that, on a white background, melanophore size was reduced first and this was followed by a decrease in melanophore density caused by gradual cell death. The process of cell death included loss of cell activity, cell fragmentation, phagocytosis of the fragments, and clearance via the epidermis. Apoptosis was assessed by the appearance of phosphatidylserine on the cell surface of melanophores that had lost motile activity, and DNA fragments involved in cell fragmentation were detected by the TUNEL (TDT-mediated dUTP-biotin nick end-labeling) assay. However, when chemically denervated fish were used, although melanophore size was reduced as expected, cell death was suppressed even on a white background. In skin tissue culture, apoptosis in melanophores was stimulated significantly by norepinephrine, but not by melanin-concentrating hormone. These results indicate that melanophore density decreases by apoptosis, and suggest that sympathetic innervation has an important role in the regulation of apoptosis in melanophores. In analogous fashion, leucophores showed a significant decrease in density with an increase of cell death on a black background. We suggest that apoptosis regulates the balance of pigment cells in the skin of medaka fish to adapt their body color to a particular background.     

Identification and characterization of Thermus thermophilus HB8 RuvA protein, the subunit of the RuvAB protein complex that promotes branch migration of Holliday junctions

The Escherichia coli ruvA and ruvB genes constitute an SOS-regulated operon. The products of these genes form a protein complex that promotes branch migration of the Holliday junction, an intermediate of homologous recombination. RuvA protein binds specifically to the Holliday junction and recruits RuvB protein to the junction. RuvB is an ATP-driven motor protein involved in branch migration. We previously cloned the ruvB gene of the thermophilic bacterium Thermus thermophilus HB8 (Tth) and found that, in contrast to the operon structure in most mesothermic bacteria, the ruvA gene is absent from the vicinity of ruvB. In this work, we cloned the ruvA gene from T. thermophilus HB8 and analyzed its nucleotide sequence. Tth RuvA is a protein of 20,414 Da consisting of 191 amino acid residues, and is 37% identical in amino acid sequence to E. coli RuvA. Tth ruvA complemented the DNA repair defect of E. coli deltaruvA mutants. The purified Tth RuvA protein stimulated Tth RuvB activities, such as hydrolysis of ATP and promotion of branch migration of the Holliday junction, in a manner similar to the RuvA-RuvB interactions observed in E. coli. In addition, Tth RuvA stimulated the E. coli RuvB activities in vitro, which was well consistent with the results of in vivo hetero-complementation experiments.     

Dynamics of the cluster model of protein folding

The cluster model of protein folding [Kanehisa, M. I. & Tsong, T. Y. (1978) J. Mol. Biol. 124 , 177–194] is further investigated for the thermodynamic and kinetic properties of protein folding–unfolding transitions. A cluster is a locally formed ordered region in the polypeptide chain due to cooperative interactions among residues. In the cluster model a cooperative term is assigned as proportional to the surface area of a globular cluster. This assignment is compared with that for the helix–coil transition of homopolypeptides, where the cooperative term is proportional to the two ends of a linear helical sequence. The dynamics of the cluster model exhibit a slow phase, which is well-separated from other faster phases, because of the cooperative interaction of the macrosystem. This slow phase not only appears within the transition region, but can also persist well below the transition region if the cooperativity depends on the external condition. The amplitudes of certain kinetic phases can vary depending on the choice of physical parameters monitoring the reaction. Thus the same reaction may display different time courses. The qualitative aspects of the folding dynamics are as follows. In one case the rate-limiting formation of a critical-size cluster is followed by its rapid growth, while in the other the rate-limiting step appears in a later stage, where preformed smaller clusters merge into larger ones. The former case is similar to the dynamics of the helix–coil transition, and the latter represents a stepwise mechanism of protein structure formation.     

Long-lasting change in brain dynamics induced by methamphetamine: enhancement of protein kinase C-dependent astrocytic response and behavioral sensitization

It is well known that long-term exposure to psychostimulants induces neuronal plasticity. Recently, accumulating evidence suggests that astrocytes may actively participate in synaptic plasticity. In this study, we found that in vitro treatment of cortical neuron/glia co-cultures with either methamphetamine (METH) or morphine (MRP) caused the activation of astrocytes via protein kinase C (PKC). Purified astrocytes were markedly activated by METH, whereas MRP had no such effect. METH, but not MRP, caused a long-lasting astrocytic activation in cortical neuron/glia co-cultures. Furthermore, MRP-induced behavioral sensitization to hyper-locomotion was reversed by 2 months of withdrawal following intermitted MRP administration, whereas behavioral sensitization to METH-induced hyper-locomotion was maintained even after 2 months of withdrawal. Consistent with this cell culture study, in vivo treatment with METH, which was associated with behavioral sensitization, caused a PKC-dependent astrocytic activation in the cingulate cortex and nucleus accumbens of mice. These findings provide direct evidence that METH induces a long-lasting astrocytic activation and behavioral sensitization through the stimulation of PKC in the rodent brain. In contrast, MRP produced a reversible activation of astrocytes via neuronal PKC and a reversibility of behavioral sensitization. This information can break through the definition of drugs of abuse and the misleading of concept that morphine produces a long-lasting neurotoxicity.     

Administration of ANG II induces iron deposition and upregulation of TGF-beta1 mRNA in the rat liver

We previously found that ANG II infusion into rats causes iron deposition in the kidney and heart, which may have a role in the regulation of profibrotic gene expression and tissue fibrosis. In the present study, we have investigated whether ANG II can also induce iron accumulation in the liver. Prussian blue staining detected frequent iron deposition in the interstitium of the liver of rats treated with pressor dose ANG II for 7 days, whereas iron deposition was absent in the livers of control rats. Immunohistochemical and histological analyses showed that some iron-positive nonparenchymal cells were positive for ferritin and heme oxygenase-1 (HO-1) protein and TGF-beta1 mRNA and were judged to be monocytes/macrophages. It was shown that ANG II infusion caused about a fourfold increase in ferritin and HO-1 protein expression by Western blot analysis and about a twofold increase in TGF-beta1 mRNA expression by Northern blot analysis, which were both suppressed by treating ANG II-infused rats with losartan and deferoxamine. In addition, mild interstitial fibrosis was observed in the liver of rats that had been treated with pressor dose ANG II for 7 days or with nonpressor dose ANG II for 30 days, the latter of which also caused loss of hepatocytes and intrahepatic hemorrhage in the liver. Taken together, our data suggest that ANG II infusion induces aberrant iron homeostasis in the liver, which may have a role in the ANG II-induced upregulation of profibrotic gene expression in the liver.     

Inhibitory effects of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) on food intake in the goldfish, Carassius auratus

Pituitary adenylate cyclase-activating polypeptide (PACAP) has a similar structure to that of vasoactive intestinal peptide (VIP) and both the polypeptides belong to the same molecular group, the secretin-glucagon superfamily. PACAP and VIP have possible potency as hypothalamic factors mediating the release of pituitary hormones in the fish pituitary. However, the roles of PACAP and VIP in the central nervous systems of fish have not yet been made clear. Recently, it was reported that PACAP and/or VIP are involved in the feeding behavior of the mouse and chick. Therefore, we investigated the effects of intracerebroventricular (ICV) and intraperitoneal (IP) administration of synthetic PACAP and VIP on food intake in the goldfish, Carassius auratus. Cumulative food intake was significantly decreased by ICV injection of PACAP (11 or 22 pmol/g body weight) or VIP (11 or 22 pmol/g) during a 60-min observation period after treatment. IP administration of PACAP (44 or 88 pmol/g) or VIP (22 or 44 pmol/g) induced a significant decrease in food intake during a 60-min observation period after treatment. These results suggest that PACAP and VIP may be involved as feeding regulators in goldfish.