Enrichment and characterization of a bacterial mixture capable of utilizing <Emphasis Type="Italic">C</Emphasis>-mannosyl tryptophan as a carbon source

C-Mannosylation, a protein-modification found in various eukaryotes, involves the attachment of a single mannose molecule to selected tryptophan residues of proteins. Since C-mannosyl tryptophan (CMW) was detected in human urine, it is generally thought that CMW is not catabolized inside our body and instead is excreted via the urine. This paper reports enrichment of a bacterial consortium from soil that degrades CMW. The bacteria grew in minimal medium supplemented with CMW as the carbon source. Interestingly, even after successive clonal picks of individual colonies, several species were still present in each colony as revealed by 16S rRNA gene sequence analysis, indicating that a single species may not be responsible for this activity. A next generation sequencing (NGS) analysis was therefore carried out in order to determine which bacteria were responsible for the catabolism of CMW. It was found that a species of Sphingomonadaceae family, but not others, increased with simultaneous decrease of CMW in the media, suggesting that this species is most likely the one that is actively involved in the degradation of CMW.     

Purification and properties of beef liver aldehyde reductase catalyzing the reduction of D-erythrose 4-phosphate

An aldehyde reductase catalyzing the NADPH-dependent reduction of D-erythrose 4-phosphate to D-erythritol 4-phosphate was purified from beef liver. It was proved to be homogeneous by polyacrylamide gel electrophoresis, sodium dodecyl sulfate polyacrylamide gel electrophoresis and ultracentrifugation analysis. The enzyme was proved to be a monomeric enzyme and its molecular weight was about 40,000. The enzyme was able to reduce not only tetroses but also trioses, aromatic aldehydes, D-glucuronate and succinic semialdehyde. Apparent Km-values for aromatic aldehydes were lower than those for tetroses, trioses, D-glucuronate and succinic semi-aldehyde. Barbiturates and valproate were potent inhibitors of the enzyme and their apparent K1-values were in the range of 80-180 microM. Quercitrin was the most potent inhibitor and its K1-value was about 7 microM. From the viewpoint of substrate specificity and inhibitor sensitivity, it seems that the enzyme belongs to the high-Km type aldehyde reductases.     

Protection against Leishmania major infection by oligomannose-coated liposomes

Liposomes coated with neoglycolipids constructed with mannopentaose and dipalmitoylphosphatidylethanolamine (Man5-DPPE) have been shown to induce cellular immunity against antigens encapsulated in the liposomes. To assess whether these neoglycolipid-coated liposomes can elicit protective immune response against challenge infection, effects of immunization with soluble leishmanial antigens encapsulated in the liposomes were evaluated using Leishmania major infection in susceptible BALB/c mice. Intraperitoneal immunization of mice with leishmanial antigens in the Man5-DPPE-coated liposomes significantly suppressed footpad swelling in comparison to the control, non-immunized mice, while progression of the disease was observed in mice administered antigens in uncoated liposomes and those administered soluble antigens alone, as seen with control mice. Similarly, the number of parasites decreased substantially in local lymph nodes of mice immunized with the antigen in the Man5-DPPE-coated liposomes. Protection against L. major infection in the immunized mice also coincided with an elevated ratio of antigen-specific IgG2a/IgG1 antibodies, which is a profile of T helper-type 1-like immune response. Taken together, these results indicate the possibility that Man5-DPPE-coated liposome-encapsulated antigens could serve as a vaccine that triggers protection against infectious disease.     

Mechanical stretch augments insulin-induced vascular smooth muscle cell proliferation by insulin-like growth factor-1 receptor

Insulin resistance and hypertension have been implicated in the pathogenesis of cardiovascular disease; however, little is known about the roles of insulin and mechanical force in vascular smooth muscle cell (VSMC) remodeling. We investigated the contribution of mechanical stretch to insulin-induced VSMC proliferation. Thymidine incorporation was stimulated by insulin in stretched VSMCs, but not in un-stretched VSMCs. Insulin increased 2-deoxy-glucose incorporation in both stretched and un-stretched VSMCs. Mechanical stretch augmented insulin-induced extracellular signal-regulated kinase (ERK) and Akt phosphorylation. Inhibitors of epidermal growth factor (EGF) receptor tyrosine kinase and Src attenuated insulin-induced ERK and Akt phosphorylation, as well as thymidine incorporation, whereas 2-deoxy-glucose incorporation was not affected by these inhibitors. Moreover, stretch augmented insulin-like growth factor (IGF)-1 receptor expression, although it did not alter the expression of insulin receptor and insulin receptor substrate-1. Insulin-induced ERK and Akt activation, and thymidine incorporation were inhibited by siRNA for the IGF-1 receptor. Mechanical stretch augments insulin-induced VSMC proliferation via upregulation of IGF-1 receptor, and downstream Src/EGF receptor-mediated ERK and Akt activation. Similar to in vitro experiment, IGF-1 receptor expression was also augmented in hypertensive rats. These results provide a basis for clarifying the molecular mechanisms of vascular remodeling in hypertensive patients with hyperinsulinemia.     

Control of Simian Immunodeficiency Virus Replication by Vaccine-Induced Gag- and Vif-Specific CD8+ T Cells

For development of an effective T cell-based AIDS vaccine, it is critical to define the antigens that elicit the most potent responses. Recent studies have suggested that Gag-specific and possibly Vif/Nef-specific CD8⁺ T cells can be important in control of the AIDS virus. Here, we tested whether induction of these CD8⁺ T cells by prophylactic vaccination can result in control of simian immunodeficiency virus (SIV) replication in Burmese rhesus macaques sharing the major histocompatibility complex class I (MHC-I) haplotype 90-010-Ie associated with dominant Nef-specific CD8⁺ T-cell responses. In the first group vaccinated with Gag-expressing vectors (n = 5 animals), three animals that showed efficient Gag-specific CD8⁺ T-cell responses in the acute phase postchallenge controlled SIV replication. In the second group vaccinated with Vif- and Nef-expressing vectors (n = 6 animals), three animals that elicited Vif-specific CD8⁺ T-cell responses in the acute phase showed SIV control, whereas the remaining three with Nef-specific but not Vif-specific CD8⁺ T-cell responses failed to control SIV replication. Analysis of 18 animals, consisting of seven unvaccinated noncontrollers and the 11 vaccinees described above, revealed that the sum of Gag- and Vif-specific CD8⁺ T-cell frequencies in the acute phase was inversely correlated with plasma viral loads in the chronic phase. Our results suggest that replication of the AIDS virus can be controlled by vaccine-induced subdominant Gag/Vif epitope-specific CD8⁺ T cells, providing a rationale for the induction of Gag- and/or Vif-specific CD8⁺ T-cell responses by prophylactic AIDS vaccines.     

Stimulation of Expression of a Silica-Induced Protein (Sip) in Thermus thermophilus by Supersaturated Silicic Acid

The effects of silicic acid on the growth of Thermus thermophilus TMY, an extreme thermophile isolated from a siliceous deposit formed from geothermal water at a geothermal power plant in Japan, were examined at 75°C. At concentrations higher than the solubility of amorphous silica (400 to 700 ppm SiO₂), a silica-induced protein (Sip) was isolated from the cell envelope fraction of log-phase TMY cells grown in the presence of supersaturated silicic acid. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the molecular mass and pI of Sip to be about 35 kDa and 9.5, respectively. Induction of Sip expression occurred within 1 h after the addition of a supersaturating concentration of silicic acid to TM broth. Expression of Sip-like proteins was also observed in other thermophiles, including T. thermophilus HB8 and Thermus aquaticus YT-1. The amino acid sequence of Sip was similar to that of the predicted solute-binding protein of the Fe³⁺ ABC transporter in T. thermophilus HB8 (locus tag, TTHA1628; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"NC_006461","term_id":"55979969","term_text":"NC_006461"}}NC_006461; GeneID, 3169376). The sip gene (987-bp) product showed 87% identity with the TTHA1628 product and the presumed Fe³⁺-binding protein of T. thermophilus HB27 (locus tag TTC1264; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"NC_005835","term_id":"46198308","term_text":"NC_005835"}}NC_005835; GeneID, 2774619). Within the genome, sip is situated as a component of the Fbp-type ABC transporter operon, which contains a palindromic structure immediately downstream of sip. This structure is conserved in other T. thermophilus genomes and may function as a terminator that causes definitive Sip expression in response to silica stress.Occurring mainly in the form of silica (SiO₂), silicon (Si) is the second-most abundant element in the earth''s crust, accounting for 28.8% of the earth''s mass (34). SiO₂ exists as monosilicic acid (Si(OH)₄) in aqueous solution, as represented in the following equation: SiO₂ + 2H₂O · (Si(OH)₄). The solubility of silica greatly depends on temperature, pH (17), and salt concentration, among other parameters (27). As the temperature of a silicic acid solution declines, its concentration can exceed the solubility of amorphous silica. Under those conditions, silicic acid polymerizes to form polysilisic acid, which is relatively stable in aqueous solution because the repulsion between the negative charges on its surface keeps it from readily aggregating and precipitating. In a geothermal reservoir, at high temperature and pressure, the silicic acid concentration at equilibrium shows the solubility of quartz. However, when that geothermal water is discharged to the surface, the silicic acid concentration becomes supersaturated as the water boils, frequently leading to the formation of siliceous deposits called “silica sinter” (11). Microscopic observation of such siliceous deposits reveals many microbe-like structures (20), and it has been suggested that these fossils represent archean microorganisms that grew in the hot, supersaturated fluids (26). There have been a number of experimental studies carried out with the aim of characterizing the physical changes associated with various bacteria during silicification (23, 26, 31, 33, 37); however, the effect of silica on the bacterial habitat in geothermal environments and the mechanism by which siliceous deposits are formed remain unexplained.Recent studies have shown that biosilicification in geothermal areas reflects the activities of various thermophilic microorganisms (15, 23, 29). For instance, geothermal water and the water discharged from hydrothermal vents contain a high concentration of silicic acid, and biogenic textures covered with amorphous silica have been found in areas around both sources (12, 22). Moreover, in a study of experimental silicification, interactions were observed between silica and Sulfurihydrogenibium azorense, a representative member of the order Aquificales (26). Still, the effect of silica on the bacterial habitat in thermal environments and the molecular properties affecting aggregation and siliceous deposition remain poorly understood.Our previous studies have focused on the effect of bacteria on the formation of siliceous deposits in geothermal water (18, 21). Siliceous deposits (called silica scale) that form in pipelines and on surface equipment in geothermal power plants cause serious economic problems related to energy loss and to plant maintenance throughout the world (38). We also observed that a Thermus strain isolated from silica scale (TMY) was able to efficiently generate and deposit amorphous silica in vitro, beginning in the latter part of the log growth phase (19). On the basis of its morphological, physiological, and genetic properties, the organism was identified as a strain of Thermus thermophilus, and its distinct properties were indicative of the microdiversity of T. thermophilus strains (14). Thermus strains are the predominant heterotrophs in natural geothermal and hydrothermal habitats, and thus far, the genomes of T. thermophilus strains HB8 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AP008226","term_id":"55771382","term_text":"AP008226"}}AP008226) and HB27 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AE017221","term_id":"46197919","term_text":"AE017221"}}AE017221) have been sequenced completely (16). Given its genomic similarity to T. thermophilus HB8, we analyzed the genetic information of strain TMY in that context.Here we report the effect of silicic acid concentration on the growth of T. thermophilus TMY, which was isolated from a siliceous deposit formed at a geothermal electric power plant. Notably, supersaturated silicic acid markedly stimulated expression of one cell envelope protein, which we named silica-induced protein (Sip). Induction of Sip expression occurred rapidly after the cells were exposed to supersaturated silicic acid, and the amino acid sequence of Sip showed significant similarity with the Fe³⁺ ABC transporters observed in other Thermus strains. These results shed new light on the growth and biosilisification associated with thermophiles in geothermal environments.     

Antimicrobial Browning-Inhibitory Effect of Flavor Compounds in Seaweeds

Since ancient times, the antimicrobial properties of seaweeds have been recognized. However, antimicrobial activities of volatile compounds in seaweeds have not been explored so far. Here, essential oils from seaweeds including green, brown and red algae such as Laminaria japonica, Kjellmaniella crassifolia, Gracilaria verrucosa and Ulva pertusa were prepared by using SDE (simultaneous distillation and extraction) apparatus. Volatile compounds in the essential oils were identified as aldehydes, ketones, carboxylic acids, alcohols and hydrocarbons by comparison of GC-retention times and MS data with those of authentic specimens. Flavor compounds such as (3Z)-hexenal, (2E)-hexenal and (2E)-nonenal in some essential oils showed strong antimicrobial activities against Escherichia coli TG-1, and Erwinia carotovora. Inhibition of browning can be achieved during either of two stages, namely, oxidation reaction by tyrosinase or subsequent non-enzymatic polymerization. Tyrosinase activity was measured by monitoring absorbance at 475 nm originating from dopachrome formed from L-DOPA. Many kinds of aliphatic carboxylic acids, aldehydes and alcohols were used as inhibitors for PPO activity. The results indicated that the α,β-unsaturated carbonyl compounds strongly inhibit tyrosinase activity. When seaweeds are damaged or macerated, the α,β-unsaturated aldehydes such as (2E)-hexenal and (2E)-nonenal are biosynthesized via the corresponding (3Z)-unsaturated aldehydes from linolenic acid and arachidonic acid. The flavor compounds that are formed could be valuable as safe antimicrobial browning-inhibitory agents of edible seaweed origin.