First systematic chiral syntheses of two pairs of enantiomers with 3,5-dihydroxyheptenoic acid chain, associated with a potent synthetic statin NK-104

First systematic chiral syntheses of two pairs of enantiomers with 3,5-dihydroxyheptenoic acid chain, associated with a potent synthetic statin NK-104 are reported. A pair of syn diol isomers (NK-104 and its enantiomer) was obtained efficiently by diastereomeric resolution. The synthesis of a pair of anti diol isomers (3-epimer and 5-epimer) was accomplished effectively by the asymmetric aldol reaction followed by anti stereoselective reduction as key steps. Their purity determinations were effected by chiral HPLC analysis.     

Enzymatic synthesis of a novel glycolipid biosurfactant, mannosylerythritol lipid-D and its aqueous phase behavior

Mannosylerythritol lipids (MELs) produced by yeasts are one of the most promising glycolipid biosurfactants. In this study, we succeeded in the preparation of a novel MEL homolog having no acetyl groups, namely MEL-D. MEL-D was synthesized by lipase-catalyzed hydrolysis of acetyl groups from a known MEL, and identified as 4-O-[2′,3′-di-O-alka(e)noyl-β-d-mannopyranosyl]-(2R,3S)-erythritol. The obtained MEL-D showed a higher critical aggregation concentration (CAC = 1.2 × 10⁻⁵ M) and hydrophilicity compared to known MELs, retaining an excellent surface tension lowering activity (the surface tension at the CAC was 24.5 mN/m). In addition, we estimated the binary phase diagram of the MEL-D–water system based on a combination of visual inspection, polarized optical microscopy, and SAXS measurement. From these results, MEL-D was found to self-assemble into a lamellar (L_α) structure over all ranges of concentration. Meanwhile, the one-phase L_α region of MEL-D was extended wider than those of known MELs. MEL-D might keep more water between the polar layers in accordance with the extension of the interlayer spacing (d). These results suggest that the newly obtained MEL-D would facilitate the application of MELs in various fields as a lamellar-forming glycolipid with higher hydrate ability.     

Early Staphylococcal Biofilm Formation on Solid Orthopaedic Implant Materials: In Vitro Study

Biofilms forming on the surface of biomaterials can cause intractable implant-related infections. Bacterial adherence and early biofilm formation are influenced by the type of biomaterial used and the physical characteristics of implant surface. In this in vitro research, we evaluated the ability of Staphylococcus epidermidis, the main pathogen in implant-related infections, to form biofilms on the surface of the solid orthopaedic biomaterials, oxidized zirconium-niobium alloy, cobalt-chromium-molybdenum alloy (Co-Cr-Mo), titanium alloy (Ti-6Al-4V), commercially pure titanium (cp-Ti) and stainless steel. A bacterial suspension of Staphylococcus epidermidis strain RP62A (ATCC35984) was added to the surface of specimens and incubated. The stained biofilms were imaged with a digital optical microscope and the biofilm coverage rate (BCR) was calculated. The total amount of biofilm was determined with the crystal violet assay and the number of viable cells in the biofilm was counted using the plate count method. The BCR of all the biomaterials rose in proportion to culture duration. After culturing for 2–4 hours, the BCR was similar for all materials. However, after culturing for 6 hours, the BCR for Co-Cr-Mo alloy was significantly lower than for Ti-6Al-4V, cp-Ti and stainless steel (P<0.05). The absorbance value determined in the crystal violet assay and the number of viable cells on Co-Cr-Mo were not significantly lower than for the other materials (P>0.05). These results suggest that surface properties, such as hydrophobicity or the low surface free energy of Co-Cr-Mo, may have some influence in inhibiting or delaying the two-dimensional expansion of biofilm on surfaces with a similar degree of smoothness.     

Sodium/Proton Exchange in Cultured Bovine Adrenal Medullary Cells

We investigated the presence of Na+/H+ exchange in cultured bovine adrenal medullary cells. The intracellular pH in control cells measured by 5,5-dimethyl[2-14C]oxazolidine-2,4-dione was 7.13 +/- 0.02 (n = 6). Removal of Na+ from the incubation medium shifted the intracellular pH down to 6.67 +/- 0.12 (n = 6). Reintroduction of Na+ to the medium caused a rapid recovery in intracellular pH to 7.20-7.30 that was associated with an increase in uptake of 22Na+ by the cells. Both increases in intracellular pH and uptake of 22Na+ were inhibited by amiloride, an inhibitor of Na+/H+ exchange. The recovery of intracellular pH by addition of Na+ was partially inhibited by quinidine, another inhibitor of Na+/H+ exchange, but not by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, an anion-exchange (Cl-/HCO3-) inhibitor. Li+ could substitute for Na+ in the recovery of intracellular pH. Carbachol caused an increase in intracellular pH from 7.12 +/- 0.01 to 7.21 +/- 0.02 (n = 10). This increase in intracellular pH caused by carbachol was inhibited by amiloride. These results suggest the existence of an amiloride-sensitive Na+/H+ exchange that regulates the intracellular pH in adrenal medullary cells.     

Thermostable DNA helicase improves the sensitivity of digital PCR

DNA/RNA helicases, which catalyze the unwinding of duplex nucleic acids using the energy of ATP hydrolysis, contribute to various biological functions involving DNA or RNA. Euryarchaeota-specific helicase Tk-EshA (superfamily 2) from the hyperthermophilic archaeon Thermococcus kodakarensis has been used to decrease generation of mis-amplified products (noise DNAs) during PCR. In this study, we focused on another type (superfamily 1B) of helicase, Tk-Upf1 (TK0178) from T. kodakarensis, and compared its effectiveness in PCR and digital PCR with that of Tk-EshA. For this purpose, we obtained Tk-Upf1 as a recombinant protein and assessed its enzymatic characteristics. Among various double-stranded DNA (dsDNA) substrates (forked, 5′ overhung, 3′ overhung, and blunt-ended duplex), Tk-Upf1 had the highest unwinding activity toward 5′ overhung DNAs. Noise DNAs were also eliminated in the presence of Tk-Upf1 at concentrations 10-fold lower than those required to yield a comparable reduction with Tk-EshA. When a 5′ or 3′ overhung mis-annealed primer was included as a competitive primer along with specific primers, noise DNAs derived from the mis-annealed primer were eliminated in the presence of Tk-Upf1. In digital PCR, addition of Tk-EshA or Tk-Upf1 increased fluorescent intensities and improved separation between common and risk allele clusters, indicating that both helicases functioned as signal enhancers. In comparison with Tk-EshA, a smaller amount of Tk-Upf1 was required to improve the performance of digital PCR.     

Characterization of Leptospira species isolated from soil collected in Japan

Leptospira were isolated from soil obtained from Hokkaido, the northernmost island, to Okinawa, the southernmost island, of Japan using sulfamethoxazole, trimethoprim, amphotericin B, fosfomycin, and 5‐ fluorouracil. Fifty of 132 soil samples (37.9%) were culture‐positive. On the basis of 16S‐rDNA sequences, 12 of the isolated Leptospira were classified into a pathogenic species clade that is closely associated with L. alstonii and L. kmetyi. Nine isolates were classified as intermediate species and were found to be similar to L. licerasiae. Twenty‐seven isolates were classified as non‐pathogenic species, of which 23 were found to be related to L. wolbachii. Non‐pathogenic Leptospira are commonly distributed in environmental soil.     

Rapid and Sensitive PCR Detection of Vibrio trachuri Pathogenic to Japanese Horse Mackerel (Trachurus japonicus)

A polymerase chain reaction (PCR) method was performed for rapid and sensitive detection of pathogenic Vibrio trachuri isolated from cultured Japanese horse mackerel. A set of primers was selected from the base sequence of the Pst I fragment of T9210 chromosomal DNA and used for PCR detection of T9210. This PCR specifically amplified the DNAs from V. trachuri T9210, T9213, and T9216 but not of those other bacterial strains. PCR using a Pst I-1 primer set made it possible to detect 100 fg of T9210 DNA. The PCR method reported here may be useful for detection and identification of V. trachuri pathogenic to Japanese horse mackerel.