Isolation and characterization of fructophilic lactic acid bacteria from fructose-rich niches

Fourteen strains of fructophilic lactic acid bacteria were isolated from fructose-rich niches, flowers, and fruits. Phylogenetic analysis and BLAST analysis of 16S rDNA sequences identified six strains as Lactobacillus kunkeei, four as Fructobacillus pseudoficulneus, and one as Fructobacillus fructosus. The remaining three strains grouped within the Lactobacillus buchneri phylogenetic subcluster, but shared low sequence similarities to other known Lactobacillus spp. The fructophilic strains fermented only a few carbohydrates and fermented d-fructose faster than d-glucose. Based on the growth characteristics, the 14 isolates were divided into two groups. Strains in the first group containing L. kunkeei, F. fructosus, and F. pseudoficulneus grew well on d-fructose and on d-glucose with pyruvate or oxygen as external electron acceptors, but poorly on d-glucose without the electron acceptors. Strains in this group were classified as “obligately” fructophilic lactic acid bacteria. The second group contained three unidentified strains of Lactobacillus that grew well on d-fructose and on d-glucose with the electron acceptors. These strains grew on d-glucose without the electron acceptors, but at a delayed rate. Strains in this group were classified as facultatively fructophilic lactic acid bacteria. All fructophilic isolates were heterofermentative lactic acid bacteria, but “obligately” fructophilic lactic acid bacteria mainly produced lactic acid and acetic acid and very little ethanol from d-glucose. Facultatively fructophilic strains produced lactic acid, acetic acid and ethanol, but at a ratio different from that recorded for heterofermentative lactic acid bacteria. These unique characteristics may have been obtained through adaptation to the habitat.     

The Aromatic Ring of Phenylalanine 334 Is Essential for Oligomerization of Vibrio vulnificus Hemolysin

Vibrio vulnificus hemolysin (VVH) is thought to be a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins. To date, the structure-function relationships of CDCs produced by Gram-negative bacteria remain largely unknown. We show here that the aromatic ring of phenylalanine residue conserved in Vibrionaceae hemolysins is essential for oligomerization of VVH. We generated the VVH mutants; substituted Phe 334 for Ile (F334I), Ala (F334A), Tyr (F334Y), or Trp (F334W); and tested their binding and oligomerizing activity on Chinese hamster ovary cells. Binding in all mutants fell by approximately 50% compared with that in the wild type. Oligomerizing activities were completely eliminated in F334I and F334A mutants, whereas this ability was partially retained in F334Y and F334W mutants. These findings indicate that both hydrophobicity and an aromatic ring residue at the 334th position were needed for full binding activity and that the oligomerizing activity of this toxin was dependent on the existence of an aromatic ring residue at the 334th position. Our findings might help further understanding of the structure-and-function relationships in Vibrionaceae hemolysins.Vibrio vulnificus hemolysin (VVH) is a pore-forming toxin produced by the Gram-negative bacterium Vibrio vulnificus (6, 11). VVH binds directly to cholesterol and is oligomerized in vitro. Once VVH forms the VVH-cholesterol complex, it can no longer bind to susceptible cells (10). Therefore, VVH could be considered a member of the cholesterol-dependent cytolysin (CDC) toxin family (35).A wide variety of Gram-positive and some Gram-negative bacteria produce CDCs, which require cellular cholesterol to exert their cytotoxicity (22, 38). Structure-function relationships between CDCs produced by Gram-positive bacteria (gpCDCs) have been studied intensively for over a decade, whereas CDCs produced by Gram-negative bacteria remain largely unknown. On the other hand, it is well known that some Vibrionaceae bacteria, such as Vibrio vulnificus, Vibrio cholerae, Aeromonas hydrophila, and Aeromonas sobria, produce pore-forming toxins/hemolysins. Among them, it was reported that VVH and Vibrio cholerae cytolysin (VCC) required cholesterol to exert their activity (12, 35). Thus, Vibrionaceae hemolysins are thought to be members of the CDC family. The generalized toxic steps are thought to be similar for both gpCDCs and Vibrionaceae hemolysins (22); i.e., monomers interact with a susceptible cell membrane, these monomers are assembled to form oligomers by membrane fluidity, and transmembrane pore formation follows (5, 22, 27, 30, 37). Although gpCDCs and Vibrionaceae hemolysins have common toxic steps, the following differences exist between them. (i) There is no similarity in amino acid sequences. (ii) gpCDCs have a highly conserved tryptophan-rich motif, which is involved in membrane recognition (3, 9, 27), whereas this motif does not exist in Vibrionaceae hemolysins. (iii) gpCDCs, such as perfringolysin and intermedilicine, are composed of four domains, whereas Vibrionaceae hemolysins are composed of two or three domains (21, 24, 25). (iv) Vibrionaceae hemolysins form pores that are smaller (2 to 3 nm in diameter) (33, 36) than those formed by gpCDCs (approximately 30 nm) (1, 2, 19).Recently, the crystal structure of VCC was determined (21). VCC is composed of three domains, namely the cytolysin domain, the β-trefoil lectin domain, and the β-prism lectin domain (21). The proposed mechanisms of action of VCC are as follows: (i) monomer binding to cell surfaces via interactions with the cytolysin domain, (ii) binding to carbohydrate receptors by the β-prism lectin domain, (iii) oligomerization via the cytolysin domain, and (iv) pore formation by insertion of a stem-loop from the cytolysin domain into the cellular membrane (21). On the other hand, from the analysis of the VVH amino acid sequence, it has been predicted that VVH is composed of two domains (21) and is missing the β-prism lectin domain, which binds to carbohydrate receptors on the cellular membrane (21). Therefore, the structure and functions of VVH are thought to be slightly different from those of VCC. Thus, analysis of the structure-function relationship of VVH will aid in the understanding of the evolutionary process of CDCs as well as of the toxic mechanism of VVH.In this study, we show that phenylalanine in the 334th position (F334) is required for the binding and oligomerizing ability of VVH. In particular, the benzene ring of this phenylalanine is a prerequisite for its oligomerizing ability. Because of the high conservation of this phenylalanine in other Vibrionaceae hemolysins, our results will contribute to a better understanding of the structure-function relationships of Vibrionaceae hemolysins.     

Crystal Structure of Exotype Alginate Lyase Atu3025 from Agrobacterium tumefaciens

Alginate, a major component of the cell wall matrix in brown seaweeds, is degraded by alginate lyases through a β-elimination reaction. Almost all alginate lyases act endolytically on substrate, thereby yielding unsaturated oligouronic acids having 4-deoxy-l-erythro-hex-4-enepyranosyluronic acid at the nonreducing end. In contrast, Agrobacterium tumefaciens alginate lyase Atu3025, a member of polysaccharide lyase family 15, acts on alginate polysaccharides and oligosaccharides exolytically and releases unsaturated monosaccharides from the substrate terminal. The crystal structures of Atu3025 and its inactive mutant in complex with alginate trisaccharide (H531A/ΔGGG) were determined at 2.10- and 2.99-Å resolutions with final R-factors of 18.3 and 19.9%, respectively, by x-ray crystallography. The enzyme is comprised of an α/α-barrel + anti-parallel β-sheet as a basic scaffold, and its structural fold has not been seen in alginate lyases analyzed thus far. The structural analysis of H531A/ΔGGG and subsequent site-directed mutagenesis studies proposed the enzyme reaction mechanism, with His³¹¹ and Tyr³⁶⁵ as the catalytic base and acid, respectively. Two structural determinants, i.e. a short α-helix in the central α/α-barrel domain and a conformational change at the interface between the central and C-terminal domains, are essential for the exolytic mode of action. This is, to our knowledge, the first report on the structure of the family 15 enzyme.     

Effect of drug transporter genes on cysteine export and overproduction in Escherichia coli

L-cysteine is an important amino acid in terms of its industrial applications. We previously found a marked production of L-cysteine from glucose in recombinant Escherichia coli cells expressing an altered cysE gene encoding feedback inhibition-insensitive serine acetyltransferase. Also, a lower level of cysteine desulfhydrase (CD) activity, which is involved in L-cysteine degradation, increased L-cysteine productivity in E. coli. The use of an L-cysteine efflux system could be promising for breeding L-cysteine overproducers. In addition to YdeD and YfiK, which have been reported previously as L-cysteine exporter proteins in E. coli, we analyzed the effects of 33 putative drug transporter genes in E. coli on L-cysteine export and overproduction. Overexpression of the acrD, acrEF, bcr, cusA, emrAB, emrKY, ybjYZ, and yojIH genes reversed the growth inhibition of tnaA (the major CD gene)-disrupted E. coli cells by L-cysteine. We also found that overexpression of these eight genes reduces intracellular L-cysteine levels after cultivation in the presence of L-cysteine. Amino acid transport assays showed that Bcr overexpression conferring bicyclomycin and tetracycline resistance specifically promotes L-cysteine export driven by energy derived from the proton gradient. When a tnaA-disrupted E. coli strain expressing the altered cysE gene was transformed with a plasmid carrying the bcr gene, the transformant exhibited more L-cysteine production than cells carrying the vector only. A reporter gene assay suggested that the bcr gene is constitutively expressed at a substantial level. These results indicate that the multidrug transporter Bcr in the major facilitator family is involved in L-cysteine export and overproduction in genetically engineered E. coli cells.     

A novel glycoside hydrolase family 105: the structure of family 105 unsaturated rhamnogalacturonyl hydrolase complexed with a disaccharide in comparison with family 88 enzyme complexed with the disaccharide

YteR, a hypothetical protein with unknown functions, is derived from Bacillus subtilis strain 168 and has an overall structure similar to that of bacterial unsaturated glucuronyl hydrolase (UGL), although it exhibits little amino acid sequence identity with UGL. UGL releases unsaturated glucuronic acid from glycosaminoglycan treated with glycosaminoglycan lyases. The amino acid sequence of YteR shows a significant homology (26% identity) with the hypothetical protein YesR also from B. subtilis strain 168. To clarify the intrinsic functions of YteR and YesR, both proteins were overexpressed in Escherichia coli, purified, and characterized. Based on their gene arrangements in genome and enzyme properties, YteR and YesR were found to constitute a novel enzyme activity, "unsaturated rhamnogalacturonyl hydrolase," classified as new glycoside hydrolase family 105. This enzyme acts specifically on unsaturated rhamnogalacturonan (RG) obtained from RG type-I treated with RG lyases and releases an unsaturated galacturonic acid. The crystal structure of YteR complexed with unsaturated chondroitin disaccharide (UGL substrate) was obtained and compared to the structure of UGL complexed with the same disaccharide. The UGL substrate is sterically hindered with the active pocket of YteR. The protruding loop of YteR prevents the UGL substrate from being bound effectively. The most likely candidate catalytic residues for general acid/base are Asp143 in YteR and Asp135 in YesR. This is supported by three-dimensional structural and site-directed mutagenesis studies. These findings provide molecular insights into novel enzyme catalysis and sequential reaction mechanisms involved in RG-I depolymerization by bacteria.     

Two New Species of Goby of the Genus <Emphasis Type="Italic">Astrabe</Emphasis> from Japan

Two species of goby belonging to the genus Astrabe are described from Japan as new species, A. flavimaculata and A. fasciata. A. flavimaculata is distinguishable from A. lactisella, the type species and hitherto the only known species of the genus, in that it has no protrusion on the upper posterior part of the dermal fold along the upper margin of the eye, fewer scales in a longitudinal row, predorsal scales, scales on the belly, a narrower white transverse band across the base of the pectoral fins, and in life yellow markings on a dark brown ground colour except for the white transverse band across the base of the pectoral fins. A. fasciata is distinguishable from A. lactisella in that it has fewer scales in a transverse row, a narrower scaled area on the lateral side of the body, a narrower white transverse band across the base of the pectoral fins, and a white transverse band across the anterior part of the 1st dorsal fin extending to the ventral side of the body.     

Identification of membrane antigen C33 recognized by monoclonal antibodies inhibitory to human T-cell leukemia virus type 1 (HTLV-1)-induced syncytium formation: altered glycosylation of C33 antigen in HTLV-1-positive T cells.

We isolated four monoclonal antibodies (MAbs), M38, M101, M104, and C33, which were capable of inhibiting syncytium formation induced in a human T-cell line, MOLT-4-#8, by coculture with human T-cell leukemia virus type 1 (HTLV-1)-positive human T-cell lines. The MAbs had, however, no inhibitory activity on syncytium formation induced in a human osteosarcoma line, HOS, by HTLV-1-positive T-cell lines. They also did not inhibit syncytium formation induced in MOLT-4-#8 by human immunodeficiency virus type 1-positive MOLT-4. All MAbs reacted with various human cell lines of lymphoid and nonlymphoid origins, including HTLV-1-positive T-cell lines. Furthermore, they all reacted with a murine A9 clone containing human chromosome 11 fragment q23-pter. Two MAbs, M104 and C33, immunoprecipitated a membrane antigen with the same molecular size. The antigen (henceforth called C33 antigen) was about 40 to 55 kDa in HTLV-1-negative Jurkat, CEM, MOLT-4, and normal peripheral blood CD4-positive human T cells and about 40 to 75 kDa in HTLV-1-positive C91/PL, TCL-Kan, MT-2, and in fresh HTLV-1-transformed CD4-positive human T-cell lines. Pulse-chase experiments revealed that C33 antigen was synthesized as a 35-kDa precursor that was then processed to 41 to 50 kDa in MOLT-4 and to 44 to 70 kDa in C91/PL. In the presence of tunicamycin, a 28-kDa protein was synthesized. The conversion from 35 kDa to 41 to 50 kDa in MOLT-4 and to 44 to 70 kDa in C91/PL was inhibited by monensin. Treatment with N-glycanase alone, but not with sialidase and O-glycanase in combination, completely removed the sugar moiety of C33 antigen from both HTLV-1-negative Jurkat and HTLV-1-positive C91/PL. Therefore, C33 antigen has only N-linked carbohydrates, the modification of which appears to be substantially altered in the presence of the HTLV-1 genome.     

Receptor binding affinity and biological activity of C-terminal elongated forms of endothelin-1

Endothelin-1 (21 amino acids; ET-21) is considered to be derived from a precursor, proendothelin (38 amino acids; ET-38). In order to make the physiological significance of this conversion clear, we synthesized various C-terminal elongated derivatives of ET-21, such as ET-22, ET-23, ET-25, ET-31, ET-36 and ET-38 (each number implies the number of amino acid residues), and measured their receptor binding affinities and biological activities. When inhibition of [¹²⁵I]ET-21 binding to cultured rat smooth muscle cells (A10 cells) was measured, ET-21 inhibited with the highest affinity (IC₅₀ = 1.6 × 10⁻¹⁰ M) and the affinity of ET-38 was 30-fold less than that of ET-21. The binding affinities of the C-terminal elongated peptides were reduced with increasing number of amino acid residues, except for ET-22 whose affinity was lower than those of other peptides (IC₅₀ = 1.6 × 10⁻⁸ M). When contractions of rat aortic segments induced by these peptides were measured, ET-21 was the most potent (EC₅₀ = 2.8 × 10⁻¹⁰ M). All C-terminal elongated peptides, including ET-38, were more than 100-fold less active. It is noteworthy that ET-22 was the least potent peptide (EC₅₀ = 1.2 × 10⁻⁷ M). When bolus doses of C-terminal elongated peptides were administered to chemically denervated rats, the time-dependent change in blood pressure induced by each peptide was different from that induced by ET-21. Although ET-21 elicited a three phase depressor/pressor blood pressure response (an initial rapid hypotension, then a rapid transient hypertension followed by a slowly developing long-lasting hypertensive effect), the C-terminal elongated peptides, including ET-38, did not cause the initial transient hypotensive response. Very interestingly, the ability of the peptides to induce the rapid phase of hypertension in vivo does not seem to be correlated with the affinity of each peptide for the smooth muscle cell receptor, since the peptides with lower affinities for the smooth muscle receptor, such as ET-22, ET-23 and ET-25, showed more potent hypertensive effects. On the other hand, the slow and long-lasting hypertensive effect is likely to be related to the affinity of the compounds. The maximal hypertensive effects of cumulatively administered ET-21 derivatives were similar to those of ET-21. These results suggest that ET-21 is the most potent vasoconstrictor among the peptides and that the conversion from ET-38 to ET-21 may be important as an activation process.     

Effectiveness of Omeprazole-Amoxicillin-Clarithromycin (OAC) Therapy for Helicobacter pylori Infection in a Japanese Population

Background. Omeprazole or lansoprazole, amoxicillin, clarithromycin (PPI/AC) therapy has been reported to provide a high cure rate of H. pylori infection with few adverse effects. Effectiveness of H. pylori therapy may vary among different geographic regions and patient populations. However, there are few reports in Japan as to its effectiveness. We have, therefore, studied the effectiveness of H. pylori therapy in a large group of Japanese patients. Methods. For this study, 366 H. pylori-positive patients with peptic ulcer disease or non-ulcer dyspepsia (263 men and 103 women, mean age 48.5 years) were assigned to 6 groups, each receiving a different PPI/AC regimen. Group 1 received omeprazole (OPZ) 20 mg, amoxicillin (AMOX) 1500 mg, and clarithromycin (CAM) 400 mg; Group 2 OPZ 40 mg, AMOX 1500 mg, and CAM 400; and Group 3 OPZ 20 mg, AMOX 2000 mg, and CAM 600 mg daily for 14 days. The group treated with lansoprazole (LPZ) 30 mg, AMOX 1500 mg and CAM 400 mg was used for 14 days in Group 1L. OPZ 20 mg, AMOX 750 mg, and CAM 200 mg were given to Group 4 for 28 days and OPZ 20 mg, AMOX 1500 mg, CAM 400 mg was administered to Group 5 for 7 days. Cure of infection was assessed by the ¹³C urea breath test one month after completion of therapy. Results. Cure rates calculated by excluding the patients who showed borderline value of ¹³C urea breath test (Δ¹³C value between 5 and 10‰ in Groups 1, 1L, 2, 3, 4, and 5 were 82.7% (95% CI; 74–90), 88.9% (76–96), 84.9% (72–93), 81.3% (67–91), 84.6% (72–93), and 85.1% (72–94) on an intention-to-treat basis, and 88% (80–94), 95.2% (84–99), 95.6% (85– 99), 90.7% (78–97), 95.7% (85–99) and 88.9% (76–96) on a per-protocol basis, respectively. Adverse effects that affected compliance were observed in 10 of 237 patients on 14-day regiments, one of 47 on a 28-day regimen and one of 46 on a 7-day regimen. Conclusion. Two weeks PPI/AC therapy is highly effective for cure of H. pylori infection in the Japanese population. The low dose one month regimen and the one week OAC regimen were also effective in our patient population.     

Epidermal growth factor plays a crucial role in mitogenic regulation of human brain tumor stem cells

A cancer stem cell population in malignant brain tumors takes an essential part in brain tumor initiation, growth, and recurrence. Growth factors, such as epidermal growth factor, fibroblast growth factor-2, vascular endothelial growth factor, platelet-derived growth factor, and hepatocyte growth factor, are shown to support the proliferation of neural stem cells and also may play key roles in gliomagenesis. However, the responsible growth factor(s), which controls maintenance of brain tumor stem cells, is not yet uncovered. We have established three cancer stem cell lines from human gliomas. These cells were immunoreactive with the neuronal progenitor markers, nestin and CD133, and established tumors that closely resembled the features of original tumor upon transplantation into mouse brain. Three cell lines retained their self-renewal ability and proliferation only in the presence of epidermal growth factor (>2.5 ng/ml). In sharp contrast, other growth factors, including fibroblast growth factor-2, failed to support maintenance of these cells. The tyrosine kinase inhibitors of epidermal growth factor signaling (AG1478 and gefitinib) suppressed the proliferation and self-renewal of these cells. Gefitinib inhibited phosphorylation of epidermal growth factor receptor as well as Akt kinase and extracellular signal-regulated kinase 1/2. Flow cytometric analysis revealed that epidermal growth factor concentration-dependently increased the population of CD133-positive cells. Gefitinib significantly reduced CD133-positive fractions and also induced their apoptosis. These results indicate that maintenance of human brain tumor stem cells absolutely requires epidermal growth factor and that tyrosine kinase inhibitors of epidermal growth factor signaling potentially inhibit proliferation and induce apoptosis of these cells.