The subunits of Chlorobium flavocytochrome c.

The subunits of cytochrome c-553 (Chlorobium thiosulfatophilum) were studied. The cytochrome is split into a cytochrome moiety and a flavoprotein moiety by treatment with 2% trichloroacetic acid. The molecular weights of the cytochrome and flavoprotein moieties are 11,000 and 47,000, respectively. The cytochrome moiety seems to have only one cysteine residue in the molecule, although its heme appears to be quite similar to the usual heme c. The flavoprotein moiety shows absorption peaks at 350 and 452nm and is insoluble at neutral pH. When the two moieties are mixed at alkaline pH, and the pH of the mixture is then brought to neutral, the flavoprotein moiety remains soluble. However, the preparation thus obtained is different from the original cytochrome c-553.     

A Lactobacillus mutant capable of accumulating long-chain polyphosphates that enhance intestinal barrier function

Inorganic polyphosphate (polyP) was previously identified as a probiotic-derived substance that enhances intestinal barrier function. PolyP-accumulating bacteria are expected to have beneficial effects on the human gastrointestinal tract. In this study, we selected Lactobacillus paracasei JCM 1163 as a strain with the potential to accumulate polyP, because among the probiotic bacteria stored in our laboratory, it had the largest amount of polyP. The chain length of polyP accumulated in L. paracasei JCM 1163 was approximately 700 phosphate (Pi) residues. L. paracasei JCM 1163 accumulated polyP when Pi was added to Pi-starved cells. We further improved the ability of L. paracasei JCM 1163 to accumulate polyP by nitrosoguanidine mutagenesis. The mutant accumulated polyP at a level of 1500 nmol/mg protein—approximately 190 times that of the wild-type strain. PolyP extracted from the L. paracasei JCM 1163 significantly suppressed the oxidant-induced intestinal permeability in mouse small intestine. In conclusion, we have succeeded in breeding the polyP-accumulating Lactobacillus mutant that is expected to enhance intestinal barrier function.     

Epsilon-poly-L-lysine dispersity is controlled by a highly unusual nonribosomal peptide synthetase

Epsilon-Poly-L-lysine (epsilon-PL) consists of 25-35 L-lysine residues in isopeptide linkages and is one of only two amino acid homopolymers known in nature. Elucidating the biosynthetic mechanism of epsilon-PL should open new avenues for creating novel classes of biopolymers. Here we report the purification of an epsilon-PL synthetase (Pls; 130 kDa) and the cloning of its gene from an epsilon-PL-producing strain of Streptomyces albulus. Pls was found to be a membrane protein with adenylation and thiolation domains characteristic of the nonribosomal peptide synthetases (NRPSs). It had no traditional condensation or thioesterase domain; instead, it had six transmembrane domains surrounding three tandem soluble domains. These tandem domains iteratively catalyzed L-lysine polymerization using free L-lysine polymer (or monomer in the initial reaction) as acceptor and Pls-bound L-lysine as donor, directly yielding chains of diverse length. Thus, Pls is a new single-module NRPS having an amino acid ligase-like catalytic activity for peptide bond formation.     

Properties of Nucleoside Phosphorylase from Enterobacter aerogenes

The properties of uridine Phosphorylase (UPase) and purine nucleoside Phosphorylase (PNPase) at high temperature were investigated. Both enzymes were found to be distributed in a wide range of bacteria and were partially purified from Enterobacter aerogenes AJ 11125 by heat treatment, ammonium sulfate fractionation and column chromatographies onDEAE-cellulose and Sephadex G-150. The UPase was purified 109-fold, and it showed an optimum pH of 8.5 and optimum temperature of 65°C, and activity toward uridine, 2′-deoxyuridine, thymidine and uracil arabinoside but not cytidine. The Km values of UPase for uridine were 0.7 mm at 40°C and 1.8 mm at 60°C. The PNPase was purified 83-fold, and it showed an optimum pH of 6.8 and optimum temperature of 60°C, and significant activity toward purine arabinosides as well as purine ribosides. The Km values of PNPase for inosine were 0.8 mm at 40°C and 2.2 mm at 60°C.     

Mushroom tyrosinase inhibitory activity of esculetin isolated from seeds of Euphorbia lathyris L

A tyrosinase inhibitor was isolated from the seeds of Euphorbia lathyris L. by bioassay-guided fractionation and purification, using silica gel column chromatography. It was identified as esculetin by comparing its physical properties and spectral data with those of an authentic sample. The IC50 value of esculetin in the mushroom tyrosinase activity test was 43 microM. The kinetic study indicates that esculetin exhibited competitive inhibition against the oxidation of 3-(3,4-dihydroxyphenyl)-alanine by mushroom tyrosinase. The structure-activity relationships among five esculetin analogs suggest that hydroxyl groups at the C6 and C7 positions of the coumarin skeleton played an important role in the expression of tyrosinase inhibitory activity.     

Input of orexin/hypocretin neurons revealed by a genetically encoded tracer in mice

The finding of orexin/hypocretin deficiency in narcolepsy patients suggests that this hypothalamic neuropeptide plays a crucial role in regulating sleep/wakefulness states. However, very little is known about the synaptic input of orexin/hypocretin-producing neurons (orexin neurons). We applied a transgenic method to map upstream neuronal populations that have synaptic connections to orexin neurons and revealed that orexin neurons receive input from several brain areas. These include the amygdala, basal forebrain cholinergic neurons, GABAergic neurons in the preoptic area, and serotonergic neurons in the median/paramedian raphe nuclei. Monoamine-containing groups that are innervated by orexin neurons do not receive reciprocal connections, while cholinergic neurons in the basal forebrain have reciprocal connections, which might be important for consolidating wakefulness. Electrophysiological study showed that carbachol excites almost one-third of orexin neurons and inhibits a small population of orexin neurons. These neuroanatomical findings provide important insights into the neural pathways that regulate sleep/wakefulness states.     

Occurrence and subcellular location of NADH- and NADPH-linked aquacobalamin reductases in human liver

1. Both activities of NADH- and NADPH-linked aquacobalamin reductases were found in some human tissues, liver, kidney pancreas, heart, spleen, lung, cerebrum, cerebellum, adrenal glands, stomach, duodenum, jejunum, ileum, colon and bone marrow. 2. Human liver contained both enzymes with higher specific activities than any other tissues. 3. The liver NADH-linked enzyme was distributed in both mitochondrial (approx. 60%) and microsomal (40%) fractions; similar to the distribution of the NADPH-linked enzyme, but of which 40% activity was found in the mitochondria and the remaining activity was recovered in the microsomes. 4. The results suggest that the synthetic systems of the cobalamin coenzymes occur in both mitochondria and microsomes of human liver.     

Site-specific isotope labeling of long RNA for structural and mechanistic studies

A site-specific isotope labeling technique of long RNA molecules was established. This technique is comprised of two simple enzymatic reactions, namely a guanosine transfer reaction of group I self-splicing introns and a ligation with T4 DNA ligase. The trans-acting group I self-splicing intron with its external cofactor, 'isotopically labeled guanosine 5'-monophosphate' (5'-GMP), steadily gave a 5'-residue-labeled RNA fragment. This key reaction, in combination with a ligation of 5'-remainder non-labeled sequence, allowed us to prepare a site-specifically labeled RNA molecule in a high yield, and its production was confirmed with (15)N NMR spectroscopy. Such a site-specifically labeled RNA molecule can be used to detect a molecular interaction and to probe chemical features of catalytically/structurally important residues with NMR spectroscopy and possibly Raman spectroscopy and mass spectrometry.     

Identification of two novel mutations in adenine phosphoribosyltransferase gene in patients with 2,8-dihydroxyadenine urolithiasis

Five mutations in the adenine phosphoribosyltransferase (APRT) gene have been described in Japanese patients with APRT deficiency. We investigated the APRT gene from three patients with APRT deficiency and two novel mutations, G133D and V84M, were determined.