Characterization of a Degraded Product Derived from Serratia piscatorum Polysaccharide by Ultrasonication

Acidic polysaccharide, PLS F–II, was prepared from Serratia piscatorum polysaccharide, PLS N–I, by a sequence of ultrasonication and gel filtration and was examined for chemical composition and biological activity.

The purified PLS F–II preparation was shown to be homogeneous by ultracentrifugation, zone electrophoresis and column chromatography. The molecular weight was estimated to be about 2 × 10⁴ by the Archibald method. PLS F–II was composed of l-rhamnose, d-galactose and d-galacturonic acid in the molar ratio of 2: 1: 1 and was partially acylated on the galacturonic acid residues.

PLS F–II was found to enhance the antibody formation in mice, although it showed no anti-inflammatory activity.     

Adrenomedullin (11-26): a novel endogenous hypertensive peptide isolated from bovine adrenal medulla.

Adrenomedullin (AM) is a potent hypotensive peptide originally isolated from pheochromocytoma tissue. Both the ring structure and the C-terminal amide structure of AM are essential for its hypotensive activity. We have developed an RIA which recognizes the ring structure of human AM. Using this RIA, we have characterized the molecular form of AM in bovine adrenal medulla. Gel filtration chromatography revealed that three major peaks of immunoreactive AM existed in the adrenal medulla. The peptide corresponding to Mr 1500 Da was further purified to homogeneity. The peptide was determined to be AM (11-26) which has one intramolecular disulfide bond. Amino acid sequences of bovine AM and its precursor were deduced from the analyses of cDNA encoding bovine AM precursor. The synthetic AM (11-26) produced dose-dependent strong pressor responses in unanesthetized rats in vivo. The hypertensive activity lasted about one minute, and a dose dependent increase in heart rate was also observed. The present data indicate that AM (11-26) is a major component of immunoreactive AM in bovine adrenal medulla and shows pressor activity.     

Mulberry leaf powder prevents atherosclerosis in apolipoprotein E-deficient mice

Mulberry is commonly used to feed silkworms. Here we examined whether a dietary intake of mulberry leaf (ML) could affect atherogenesis in vivo and in vitro. Apolipoprotein E-deficient mice were fed either normal chow (control group) or a diet containing 1% ML powder (ML group) from 6 weeks of age. The mice were sacrificed after 12 weeks. The susceptibility of plasma lipoprotein to oxidation was assessed using diene formation. A significant increase in the lag time of lipoprotein oxidation was detected in the ML group compared with the control group. Furthermore, the ML group showed a 40% reduction in atherosclerotic lesion size in the aortae compared with the control. We also examined the direct anti-oxidative activity of ML in vitro. Aqueous extract of ML had a strong scavenging effect on 1,1-diphenyl-2-picrylhydrazyl and inhibited lipoprotein oxidation. These results confirm that ML contains anti-oxidative substances that might help prevent atherosclerosis.     

Crystal structure of the C2 domain of class II phosphatidylinositide 3-kinase C2alpha

Phosphatidylinositide (PtdIns) 3-kinase catalyzes the addition of a phosphate group to the 3'-position of phosphatidyl inositol. Accumulated evidence shows that PtdIns 3-kinase can provide a critical signal for cell proliferation, cell survival, membrane trafficking, glucose transport, and membrane ruffling. Mammalian PtdIns 3-kinases are divided into three classes based on structure and substrate specificity. A unique characteristic of class II PtdIns 3-kinases is the presence of both a phox homolog domain and a C2 domain at the C terminus. The biological function of the C2 domain of the class II PtdIns 3-kinases remains to be determined. We have determined the crystal structure of the mCPK-C2 domain, which is the first three-dimensional structural model of a C2 domain of class II PtdIns 3-kinases. Structural studies reveal that the mCPK-C2 domain has a typical anti-parallel beta-sandwich fold. Scrutiny of the surface of this C2 domain has identified three small, shallow sulfate-binding sites. On the basis of the structural features of these sulfate-binding sites, we have studied the lipid binding properties of the mCPK-C2 domain by site-directed mutagenesis. Our results show that this C2 domain binds specifically to PtdIns(3,4)P(2) and PtdIns(4,5)P(2) and that three lysine residues at SBS I site, Lys-1420, Lys-1432, and Lys-1434, are responsible for the phospholipid binding affinity.     

β-Glucuronidase from Lactobacillus brevis useful for baicalin hydrolysis belongs to glycoside hydrolase family 30

Baicalin (baicalein 7-O-β-d-glucuronide) is one of the major flavonoid glucuronides found in traditional herbal medicines. Because its aglycone, baicalein, is absorbed more quickly and shows more effective properties than baicalin, the conversion of baicalin into baicalein by β-glucuronidase (GUS) has drawn the attention of researchers. Recently, we have found that Lactobacillus brevis subsp. coagulans can convert baicalin to baicalein. Therefore, we aimed to identify and characterize the converting enzyme from L. brevis subsp. coagulans. First, we purified this enzyme from the cell-free extracts of L. brevis subsp. coagulans and cloned its gene. Surprisingly, this enzyme was found to be a GUS belonging to glycoside hydrolase (GH) family 30 (designated as LcGUS30), and its amino acid sequence has little similarity with any GUS belonging to GH families 1, 2, and 79 that have been reported so far. We then established a high-level expression and simple purification system of the recombinant LcGUS30 in Escherichia coli. The detailed analysis of the substrate specificity revealed that LcGUS30 has strict specificity toward glycon but not toward aglycones. Interestingly, LcGUS30 prefers baicalin rather than estrone 3-(β-d-glucuronide), one of the human endogenous steroid hormones. These results indicated that L. brevis subsp. coagulans and LcGUS30 should serve as powerful tools for the construction of a safe bioconversion system for baicalin. In addition, we propose that this novel type of GUS forms a new group in subfamily 3 of GH family 30.     

β<Subscript>2</Subscript>-Integrin-Mediated Adhesion and Intracellular Ca<Superscript>2+</Superscript> Release in Human Eosinophils

Human eosinophils spontaneously adhere to various substrates in the absence of exogenously added activators. In the present study a method was developed for characterizing eosinophil adhesion by measuring changes in impedance. Impedance measurements were performed in HCO₃-buffered HybriCare medium maintained in a humidified 5% CO₂ incubator at 37°C. Impedance increased by more than 1 kΩ within minutes after eosinophils made contact with the substrate, reaching a peak within 20 min. Blocking mobilization of intracellular [Ca²⁺] that precedes adhesion with BAPTA-AM (10 μM) completely inhibited the rise in impedance as well as the changes in cell shape typically observed in adherent cells. However, lowering the extracellular [Ca²⁺] with 2.5 mM EGTA did not inhibit the increase in impedance. Pretreatment with anti-CD18 antibody to block substrate interactions with β₂-integrins, or jasplakinolide (2 μM) to block actin reorganization, abolished the increase in impedance and adherent morphology of the cells. Exposure of eosinophils to the phosphatidylinositol 3 kinase inhibitor LY294002 (5 μM) or treatment with protein kinase C zeta pseudosubstrate to competitively inhibit activity of the enzyme significantly reduced the increase in impedance and inhibited the cell spreading associated with adhesion. These results demonstrate a novel method for measuring eosinophil adhesion and showed that, following formation of a tethered attachment, a rapid increase in intracellular [Ca²⁺] precedes the cytoskeletal rearrangements required for cell shape changes and plasma membrane-substrate interactions associated with adhesion.     

A Broad Distribution of the Alternative Oxidase in Microsporidian Parasites

Microsporidia are a group of obligate intracellular parasitic eukaryotes that were considered to be amitochondriate until the recent discovery of highly reduced mitochondrial organelles called mitosomes. Analysis of the complete genome of Encephalitozoon cuniculi revealed a highly reduced set of proteins in the organelle, mostly related to the assembly of iron-sulphur clusters. Oxidative phosphorylation and the Krebs cycle proteins were absent, in keeping with the notion that the microsporidia and their mitosomes are anaerobic, as is the case for other mitosome bearing eukaryotes, such as Giardia. Here we provide evidence opening the possibility that mitosomes in a number of microsporidian lineages are not completely anaerobic. Specifically, we have identified and characterized a gene encoding the alternative oxidase (AOX), a typically mitochondrial terminal oxidase in eukaryotes, in the genomes of several distantly related microsporidian species, even though this gene is absent from the complete genome of E. cuniculi. In order to confirm that these genes encode functional proteins, AOX genes from both A. locustae and T. hominis were over-expressed in E. coli and AOX activity measured spectrophotometrically using ubiquinol-1 (UQ-1) as substrate. Both A. locustae and T. hominis AOX proteins reduced UQ-1 in a cyanide and antimycin-resistant manner that was sensitive to ascofuranone, a potent inhibitor of the trypanosomal AOX. The physiological role of AOX microsporidia may be to reoxidise reducing equivalents produced by glycolysis, in a manner comparable to that observed in trypanosomes.     

Resident macrophages activated by lipopolysaccharide suppress muscle tension and initiate inflammatory response in the gastrointestinal muscle layer

A great number of macrophages is found to be evenly distributed in the muscle layer of the gastrointestinal tract. We investigated their effects on smooth muscle contraction and the initiation of immune reactions such as inflammatory responses. Macrophages were demonstrated by the uptake of FITC-dextran and their ultrastructural features were elucidated by electron microscopy. Muscle layers of rats’ ileums were incubated with lipopolysaccharide (LPS) for 4–8 h and the force of smooth muscle contraction was measured. The induction effect of inducible nitric oxide synthase (iNOS) on macrophages was then checked by immunohistochemistry. The expression of major histocompatibility complex (MHC) class II was also examined. Macrophages in the muscle layer were confirmed as resident macrophages and were different from a population of dendritic cells. After incubation with LPS, macrophages began to express iNOS and produced NO, and it reduced smooth muscle contraction. iNOS-immunopositive cells increased in a time-dependent manner. Macrophages also began to express MHC class II. The total number of macrophages did not alter after incubation. Results indicate that resident macrophages in the muscle layer induced iNOS as an inflammatory reaction, affected smooth muscle contraction, and initiated immune response in the smooth muscle layer of the gastrointestinal tract, when activated by LPS. Accepted: 24 November 1999     

The Escherichia coli cytochrome b556 gene, cybA, is assignable as sdhC in the succinate dehydrogenase gene cluster

Abstract The cytochrome b556 -deficient mutant Escherichia coli K12 strain TK3D11 [7] could not grow with succinate as the sole carbon source, but could grow well on dl -lactate. This finding suggested that cytochrome b556 is primarily responsible for oxidative metabolism and utilization of succinate. 24 Amino acid residues at the amino-terminal of purified cytochrome b556 were determined. This sequence coincided completely with amino acid residues 4 to 27, predicted from the DNA sequence of the sdhC gene, one of the unassigned open reading frames of the sdh gene cluster recently reported by Wood et al. [16]. Based on these and other results, we concluded that cybA , the gene for cytochrome b556 , is assignable as sdhC .