Serratia Protease

The substrate specificity of Serratia protease was determined using various synthetic substrates. The enzyme did not participate in the hydrolysis of di- and tri-peptides except benzoylglycylleucinamide which was split at a limited rate into hippuric acid and leucinamide. The enzyme action on larger peptides was also studied. The enzyme cleaved the gly-leu bond in eledoisin related peptide and the gly-phe bond in bradykinin. The enzyme split oxidized insulin B-chain at twelve different peptide bonds.     

Hedgehog creates a gradient of DPP activity in Drosophila wing imaginal discs

Hedgehog (HH) and Decapentaplegic (DPP) direct anteroposterior patterning in the developing Drosophila wing by functioning as short- and long-range morphogens, respectively. Here, we show that the activity of DPP is graded and is directly regulated by a novel HH-dependent mechanism. DPP activity was monitored by visualizing the activated form of Mothers against dpp (MAD), a cytoplasmic transducer of DPP signaling. We found that activated MAD levels are highest near the source of DPP but are unexpectedly low in the cells that express dpp. HH induces dpp in these cells; it also attenuates their response to DPP by downregulating expression of the DPP receptor thick veins (tkv). We suggest that regulation of tkv by HH is a key part of the mechanism that controls the level and distribution of DPP.     

GABAergic inhibition regulates developmental synapse elimination in the cerebellum

Functional neural circuit formation during development involves massive elimination of redundant synapses. In the cerebellum, one-to-one connection from excitatory climbing fiber (CF) to Purkinje cell (PC) is established by elimination of early-formed surplus CFs. This process depends on glutamatergic excitatory inputs, but contribution of GABAergic transmission remains unclear. Here, we demonstrate impaired CF synapse elimination in mouse models with diminished GABAergic transmission by mutation of a single allele for the GABA synthesizing enzyme GAD67, by conditional deletion of GAD67 from PCs and GABAergic interneurons or by pharmacological inhibition of cerebellar GAD activity. The impaired CF synapse elimination was rescued by enhancing GABA(A) receptor sensitivity in the cerebellum by locally applied diazepam. Our electrophysiological and Ca2+ imaging data suggest that GABA(A) receptor-mediated inhibition onto the PC soma from molecular layer interneurons influences CF-induced Ca2+ transients in the soma and regulates CF synapse elimination from postnatal day 10 (P10) to around P16.     

Increase in Alphaproteobacteria in association with a polychaete,Capitella sp. I,in the organically enriched sediment

We conducted bioremediation experiments on the organically enriched sediment on the sea floor just below a fish farm, introducing artificially mass-cultured colonies of deposit-feeding polychaete, Capitella sp. I. To clarify the association between the Capitella and bacteria on the efficient decomposition of the organic matter in the sediment in the experiments, we tried to identify the bacteria that increased in the microbial community in the sediment with dense patches of the Capitella. The relationship between TOC and quinone content of the sediment as an indicator of the bacterial abundance was not clear, while a significant positive correlation was found between Capitella biomass and quinone content of the sediment. In particular, ubiquinone-10, which is present in members of the class Alphaproteobacteria, increased in the sediment with dense patches of the Capitella. We performed denaturing gradient gel electrophoresis (DGGE) analyses to identify the alphaproteobacterial species in the sediment with dense patches of the worm, using two DGGE fragments obtained from the sediment samples and one fragment from the worm body. The sequences of these DGGE fragments were closely related to the specific members of the Roseobacter clade. In the associated system with the Capitella and the bacteria in the organically enriched sediment, the decomposition of the organic matter may proceed rapidly. It is very likely that the Capitella works as a promoter of bacteria in the organically enriched sediment, and feeds the increased bacteria as one of the main foods, while the bacteria decompose the organic matter in the sediment with the assistance of the Capitella.     

Bisorbicillinol inhibits Lyn tyrosine kinase for allergic response on RBL-2H3 cells

Bisorbicillinol, which is isolated from Trichoderma sp. USF2690, is an inhibitor of β-hexosaminidase release and tumor necrosis factor (TNF)-α, and Interleukin (IL)-4 secretion from rat basophilic leukemia (RBL-2H3) cells, with IC₅₀ values of 2.8?μM, 2.9?μM and 2.8?μM respectively. We showed that the inhibitory mechanism of β-hexosaminidase release and TNF-α secretion involved inhibition of Lyn, a tyrosine kinase. The inhibitory activities of bisorbicillinol indicate that this compound is a new candidate anti-allergic agent.     

Effects of Parenteral Lipid Infusion on DNA Damage in Very Low Birth Weight Infants

Very low birth weight (VLBW) infants are known to have poorly developed antioxidant system and may be at increased risk for radical damage. Previous studies have reported higher levels of lipid peroxide products in lipid emulsion used for parenteral nutrition. To examine the direct effects of parenteral lipid infusion on DNA damage in VLBW infants, we measured urinary 8-hydroxydeoxyguanosine (8-OHdG) levels in VLBW infants before, during, and after the parenteral lipid infusion. In both the lipid-infused and lipid-free groups, urinary 8-OHdG excretion levels at 14 days old were significantly ( p <0.01) lower than those at 2 and 7 days old. However, there were no significant differences in urinary 8-OHdG excretion levels between the lipid-infused and lipid-free groups at 2, 7, and 14 days old. Our results suggest that parenteral lipid infusion does not cause oxidative DNA damage, but irrespective of the infusion DNA damage during the first week of life is enhanced when compared with 14 days after birth in VLBW infants.     

Differential regulation of TRPM channels governs electrolyte homeostasis in the C. elegans intestine

The transient receptor potential (TRP) channels are implicated in various cellular processes, including sensory signal transduction and electrolyte homeostasis. We show here that the GTL-1 and GON-2 TRPM channels regulate electrolyte homeostasis in the C. elegans intestine. GON-2 is responsible for a large outwardly rectifying current of intestinal cells, and its activity is tightly regulated by intracellular Mg(2+) levels, while GTL-1 mainly contributes to appropriate Mg(2+) responsiveness of the outwardly rectifying current. We also used nickel cytotoxicity to study the function of these channels. Both GON-2 and GTL-1 are necessary for intestinal uptake of nickel, but GTL-1 is continuously active while GON-2 is inactivated at higher Mg(2+) levels. This type of differential regulation of intestinal electrolyte absorption ensures a constant supply of electrolytes through GTL-1, while occasional bursts of GON-2 activity allow rapid return to normal electrolyte concentrations following physiological perturbations.     

Characterization of the traC determinant of the Enterococcus faecalis hemolysin-bacteriocin plasmid pAD1: binding of sex pheromone.

pAD1, a conjugative, 60-kb, hemolysin-bacteriocin plasmid in Enterococcus faecalis, encodes a mating response to a small peptide sex pheromone, cAD1, secreted by potential recipient bacteria. A gene, traC, encoding a 60.7-kDa protein with a typical amino terminal signal peptide, was identified within a region that appears to encode a product that binds to exogenous pheromone. A cloned segment of DNA containing traC resulted in specific binding of cells to synthetic cAD1. The putative traC product has strong similarity to a product of the E. faecalis plasmid pCF10 as well as oligopeptide binding proteins of Escherichia coli, Salmonella typhimurium, and Bacillus subtilis.     

A long CAG repeat in the mouse Sca1 locus replicates SCA1 features and reveals the impact of protein solubility on selective neurodegeneration

A single nicotine exposure increases dopamine levels in the mesolimbic reward system for hours, but nicotine concentrations experienced by smokers desensitize nAChRs on dopamine neurons in seconds to minutes. Here, we show that persistent modulation of both GABAergic and glutamatergic synaptic transmission by nicotine can contribute to the sustained increase in dopamine neuron excitability. Nicotine enhances GABAergic transmission transiently, which is followed by a persistent depression of these inhibitory inputs due to nAChR desensitization. Simultaneously, nicotine enhances glutamatergic transmission through nAChRs that desensitize less than those on GABA neurons. The net effect is a shift toward excitation of the dopamine reward system. These results suggest that spatial and temporal differences in nicotinic receptor activity on both excitatory and inhibitory neurons in reward areas coordinate to reinforce nicotine self-administration.     

A single amino acid substitution of Leu130Ile in snake DNases I contributes to the acquisition of thermal stability.

We purified pancreatic deoxyribonucleases I (DNases I) from three snakes, Elaphe quadrivirgata, Elaphe climacophora and Agkistrodon blomhoffii, and cloned their cDNAs. Each mature snake DNase I protein comprised 262 amino acids. Wild-type snake DNases I with Leu130 were more thermally unstable than wild-type mammalian and avian DNases I with Ile130. After substitution of Leu130Ile, the thermal stabilities of the snake enzymes were higher than those of their wild-type counterparts and similar to mammalian wild-type enzyme levels. Conversely, substituting Ile130Leu of mammalian DNases I made them more thermally unstable than their wild-type counterparts. Therefore, a single amino acid substitution, Leu130Ile, might be involved in an evolutionally critical change in the thermal stabilities of vertebrate DNases I. Amphibian DNases I have a Ser205 insertion in a Ca2+-binding site of mammalian and avian enzymes that reduces their thermal stabilities [Takeshita, H., Yasuda, T., Iida, R., Nakajima, T., Mori, S., Mogi, K., Kaneko, Y. & Kishi, K. (2001) Biochem. J.357, 473-480]. Thus, it is plausible that the thermally stable wild-type DNases I of the higher vertebrates, such as mammals and birds, have been generated by a single Leu130Ile substitution of reptilian enzymes through molecular evolution following Ser205 deletion from amphibian enzymes. This mechanism may reflect one of the evolutionary changes from cold-blooded to warm-blooded vertebrates.