Effects of dexamethasone and FK506 on Helicobacter pylori-induced gastritis and bacterial viability in Mongolian gerbils

FK506 and dexamethasone were used to investigate whether or not immunosuppression affects H. pylori colonization and gastric mucosal damage induced by Helicobacter pylori in Mongolian gerbils. Two weeks after H. pylori infection, FK506 and dexamethasone or vehicle alone were subcutaneously administered once daily for the following 2 weeks. FK506 or vehicle alone was administered subcutaneously once daily for 5 weeks (1 week before and 4 weeks after infection). In H. pylori-infected animals for 4 weeks, hemorrhagic erosions and inflammatory responses (neutrophil infiltration and lymphoid follicle formation) were induced in gastric mucosa at an incidence of 100%. Both FK506 and dexamethasone administered for 2 weeks markedly reduced such mucosal changes. In these animals, H. pylori viability in the stomach was significantly elevated. FK506 administered for 5 weeks also significantly inhibited the hemorrhagic erosions, edema and neutrophil infiltration in the stomach. H. pylori viability was slightly elevated as compared with the control. It was concluded that the host immune responses might play dual roles both by deteriorating gastritis induced by H. pylori and by protecting against H. pylori infection in its early stage.     

MexAB-OprM-specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 1: discovery and early strategies for lead optimization

The identification of a series of compounds that specifically inhibit efflux by the MexAB-OprM pump system in Pseudomonas aeruginosa is described. Synthesis and in vitro structure-activity relationships (SARs) are outlined. Early leads lacked activity in animal models, and efforts to improve solubility and reduce serum protein binding by the introduction of polar groups are discussed.     

Synthesis and absolute structures of Mycoplasma pneumoniae β-glyceroglycolipid antigens

Just recently, a pair of β-glycolipids was isolated from the cell membrane of Mycoplasma pneumoniae as a mixture of the two compounds. They are the major immunodeterminants of this pathogenic Mycoplasma and indicate high medicinal potential. They have a β-(1→6)-linked disaccharide structure close to each other; one has β-d-galactopyranoside (β-Gal-type 1) at the non-reducing terminal, and another has β-d-glucopyranoside (β-Glc-type 2). In the present study, the first stereoselective synthesis was conducted for each of the two β-glycolipids 1 and 2. ¹H NMR and TLC-immunostaining studies of the synthetic compounds enable us to establish the absolute structures having the β-(1→6)-linked disaccharides at the glycerol sn-3 position.     

Functional characterization of D-galacturonic acid reductase, a key enzyme of the ascorbate biosynthesis pathway, from Euglena gracilis

D-Galacturonic acid reductase, a key enzyme in ascorbate biosynthesis, was purified to homogeneity from Euglena gracilis. The enzyme was a monomer with a molecular mass of 38-39 kDa, as judged by SDS-PAGE and gel filtration. Apparently it utilized NADPH with a Km value of 62.5+/-4.5 microM and uronic acids, such as D-galacturonic acid (Km=3.79+/-0.5 mM) and D-glucuronic acid (Km=4.67+/-0.6 mM). It failed to catalyze the reverse reaction with L-galactonic acid and NADP(+). The optimal pH for the reduction of D-galacturonic acid was 7.2. The enzyme was activated 45.6% by 0.1 mM H(2)O(2), suggesting that enzyme activity is regulated by cellular redox status. No feedback regulation of the enzyme activity by L-galactono-1,4-lactone or ascorbate was observed. N-terminal amino acid sequence analysis revealed that the enzyme is closely related to the malate dehydrogenase families.     

Effects of polyamines on two strains of Trypanosoma brucei in infected rats and in vitro culture

We studied the effects of polyamines, which are necessary for proliferation and antioxidation in Trypanosoma brucei gambiense Wellcome strain (WS) and Trypanosoma brucei brucei ILtat 1.4 strain (IL). No difference was found in activity of ornithine decarboxylase (ODC), a key enzyme in polyamine synthesis in trypanosomes, in both strains maintained in vitro; higher (P < 0.05) ODC values were found in IL in vivo. However, WS in vivo exhibited higher proliferation rates with higher spermidine content and decreased host survival times than IL. The in vitro proliferation and polyamine contents of WS increased with the addition of polyamine to the 1-difluoromethylornithine culture medium, but not IL. These results suggested that WS uses extracellular polyamine for proliferation. In the in vitro culture, WS was less tolerant of hydrogen peroxide (oxidative stress) than IL, and malondialdehyde levels in WS were higher than in IL. The expression of trypanothione synthetase mRNA in WS in vitro was higher than in IL. These results suggest that IL is dependent on the synthesis of polyamines for proliferation and reduction of oxidative stress, whereas WS is dependent on the uptake of extracellular polyamines. A thorough understanding of the differences in the metabolic capabilities of various trypanosomes is important for the design of more effective medical treatments.     

Electron microscopic study of endogenous peroxidase activity in human liver macrophages

We evaluated the conditions of fixation for ultrastructurally demonstrating the endogenous peroxidase (PO) activity of macrophages in biopsied human liver. The application of microwaving and immersion fixation with tannic acid and aldehydes allowed excellent visualization of PO activity in the nuclear envelope (NE), rough endoplasmic reticulum (rER), and cytoplasmic granules (CG), with good preservation of cellular ultrastructures. The macrophages with PO activity showed one of the following five patterns of PO localization: positive in both the NE and rER but negative in the CG (type 1); negative in both the NE and rER but positive in the CG (type 2); negative in the NE but positive in both the rER and CG (type 3); positive in all three (type 4); PO negative (type 5). The type 1 cells resembled typical Kupffer cells, type 2 cells monocytes, and type 3 and 4 cells the exudate-resident macrophages considered to be a transitional form between exudate and resident macrophages. Type 5 cells may also be a transitional form between the exudate and resident macrophage, or an end-stage macrophage derived from exudate macrophages which have lost their PO activity. Tannic-acid-aldehyde immersion fixation with microwaving may be a useful method in the study of the PO activities of macrophages in biopsied human liver specimens.     

Molecular cloning of two tandemly arranged peroxidase genes from Populus kitakamiensis and their differential regulation in the stem

A genomic library was prepared from Populus kitakamiensis and screened with the cDNA for an anionic peroxidase from P. kitakamiensis. One genomic clone was isolated that contained two tandemly oriented genes for anionic peroxidases, prxA3a and prxA4a. Both genes consisted of four exons and three introns; the introns had consensus nucleotides, namely, GT and AG, at their 5 and 3 ends, respectively. The prxA3a and prxA4a genes encoded 347 and 343 amino acid residues, respectively, including putative signal sequences at the amino-termini. Putative promoters and polyadenylation signals were found in the flanking regions of both genes. The sequence of the coding region of prxA3a was completely identical to that of the cDNA clone pA3, whereas the sequence of the coding region of prxA4a was only 73% identical to that of the cDNA clone pA3. Northern blot analysis showed that the patterns of expression of the mRNAs that corresponded to prxA3a and prxA4a differed in stems of P. kitakamiensis.     

Yeast mutant with efficient secretion identified by a novel secretory reporter, Cluc

Yeast is an important host for the production of pharmaceutical or industrial proteins by virtue of its genetic information and easy handling. A number of heterologous proteins have been produced and purified from yeast cell cultures as secreted forms. Here, we describe a novel screening system of Saccharomyces cerevisiae and its application to improve the secretion efficiency of yeast. In our system, a natural secretory luciferase from Cypridina noctiluca is used as a reporter enzyme. The accumulation of enzymatically active luciferase in culture medium makes it possible to screen many samples simultaneously in a simple and sensitive assay. Using this system, we have discovered that the deletion mutant of MON2, which encoded a scaffold protein for vesicle formation located at the late Golgi, secreted luciferase highly efficiently to the extracellular space. Thus, we conclude that this new reporter assay is useful for the improvement and screening of yeast secretory strains.