Shrimp alkaline phosphatase

Synthetic oligonucleotides were purchased from Fasmac (Kanagawa, Japan). Restriction enzymes and shrimp alkaline phosphatase were from Takara Bio (Shiga, Japan). D-luciferin potassium salt was from Nacalai Tesque (Kyoto, Japan) and the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) was from Oriental Yeast (Tokyo, Japan). Pooled human liver microsomes (HLM) prepared 50 donors, and Supersomes™ expressing CYP3A4 and NADPH-P450 reductase (CPR) were from BD Gentest (Franklin Lakes, NJ, United States). All other reagents were of the highest quality commercially available.

In vitro enzyme assays were performed as previously described [63 (link), 64 (link)]. Briefly, a 400 μL reaction system containing 400 μM IPP and 200 μM FPP (100 mM HEPES, 5 mM MgCl₂, 10 mM KCl, pH 7.5) and 0.5 μmol/L of the purified recombinant fusion protein were incubated at 30 °C for 2 h. After inoculation, the solution was mixed with 200 μL of 0.2 mol/L Tris–HCl (pH 9.5) containing bovine intestinal alkaline phosphatase (20 mg/mL, more than 10 DEA units/mg, Sigma-Aldrich) and two units of shrimp alkaline phosphatase (1 unit/μL; TaKaRa). The reaction mixture was then incubated overnight at 30 °C to hydrolyse the diphosphate products into their corresponding alcohols. The mixture was extracted with hexane, and then three parallel samples of hydrolysate were concentrated to 100 µL in the experiment. Finally, the sample products were analysed using an LC–MS system equipped with an LTQ Orbitrap XL ETD analyser (Thermo Fisher Scientific, USA). The samples were separated using an Agilent 1200 Series HPLC system at a flow rate of 250 μL/min, and the mobile phase consisted of methanol (100%). The electrospray potential was 4.5 kV in positive electrospray ionisation (ESI) mode, and the source temperature was 275 °C. Three independent cultures were analysed for each set of experiments.

The dissected tissues were subjected to DNA extraction using QIAamp DNA Mini Kit (Qiagen). A 1.4 kb region of bacterial 16S rRNA gene was amplified by PCR with the primers 10FF (5′-AGT TTG ATC ATG GCT CAG GAT-3′) (Moran et al., 2005 (link)) and 1515R (5′-GTA CGG CTA CCT TGT TAC GAC TTA G-3′) (Takiya et al., 2006 (link)) under the temperature profile of 94°C for 1 min followed by 30 cycles of 98°C for 10 s, 50°C for 15 s, and 72°C for 2 min. After checking successful PCR amplification by electrophoresis on 1.5% agarose gels, the PCR products were purified using exonuclease I (New England Biolabs) and shrimp alkaline phosphatase (Takara) at 37°C for 15 min followed by 80°C for 15 min, and sequenced as described previously (Fukatsu and Nikoh, 1998 (link)).

DNA was extracted from fresh leaves at the seedling stage using the CTAB method with minor modified [26 (link)]. SD1 Genomic DNA including exon and intron were amplified from genomic DNA using LA Taq (Takara). Polymerase chain reactions (PCR) performed with the following conditions for thermal cycling: 94°C for 4 min; then 35 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 1 min per 1000-base; followed by 72°C for seven min. All primers used for PCR and sequencing listed in S1 Table. For sequencing, 5 μL PCR product digested with five U EXOI (Biolabs) and 0.13 U Shrimp Alkaline Phosphatase (Takara) together with 1×PCR buffer and incubated at 37°C for 1 hour; the reaction was stopped by maintaining the PCR product in 80°C for 20 min. Sequencing was independently performed three times in both forward and reverse primers on ABI 3730 with BigDye terminator sequencing kits (Applied Biosystems). Sequence contigs assembled by SEQUENCHER 4.1.2 (Gene Codes Corporation).

Tissue samples were kept in CHAOS solution for at least a week to dissolve proteins at room temperature. Total DNA was extracted from the CHAOS solution with tissue samples by conventional phenol/chloroform extraction method. We used the primers reported by McFadden et al. (2006) (link) to amplify a fragment 5' end of the mitochondrial NADH-dehydrogenase subunit 2 gene (ND2) (16S647F: 5' -ACA CAG CTC GGT TTC TAT CTA CCA-3'; ND21418R: 5' -ACA TCG GGA GCC CAC ATA-3'). We also used two primers (1S: 5'-GGT ACC CTT TGT ACA CAC CGC CCG TCG CT-3'; 2SS: 5'-GCT TTG GGC GGC AGT CCC AAG CAA CCC GAC TC-3') (Wei et al. 2006 ) to amplify the internal transcribed spacer (ITS) of the nuclear ribosomal RNA gene. All PCR reactions contained 1 μL of DNA solution, 1.6 μL of 2.5 mM dNTP Mixture, 2 μL of 10X Ex Taq buffer, 2 μL of each 10 mM primer, Ex Taq (TaKaRa) 0.08 μL, and 11.32 μL of sterile distilled water. Amplifications of these markers were performed (GeneQ PCR Thermal Cycler) with the following thermal profile; 35 cycles of 90 sec at 94 °C, 60 sec at 58 °C, 60 sec at 72 °C. Amplified fragments were checked on 1% agarose gel electrophoresis. All the PCR products were subjected to digest excess primers and inactivation of dNTP using Exonuclease I (TaKaRa) and Shrimp Alkaline Phosphatase (TaKaRa). These DNA sequences were determined by ABI3000 using a research contract service (Ltd. FASMAC).

A fasting venous blood sample of 2 ml was extracted from each subject at the elbow on the morning after diagnosis and was treated with ethylenediaminetetraacetic acid dipotassium salt (EDTA-K2) to prevent coagulation. Within 1-4 h, 0.5 ml of the blood sample was added to two 1.5 ml EP tubes. Genomic DNA was extracted from 2 ml peripheral blood samples collected from each participant using a Blood Genomic DNA Extraction Kit (Tiangen, China). The SIRT1/SIRT2 genetic variants were genotyped using the SNaPshot Multiplex Kit (Genesky Biotechnologies, China). The reaction system consisted of 5 μl SNaPshot Multiplex Kit (ABI, Shanghai), 2 μl template DNA, 2 μl ddH₂O, and 1 μl primer mix. The SNaPshot response program included 95°C for 2 minutes and 94°C for 20 s (11 cycles, 65°C for 40 s and 72°C for 90 s). Then, the extension products were purified via incubation with 1 U of shrimp alkaline phosphatase (Takara, Japan) for 15 minutes at 37°C and subsequent incubation at 80°C for 15 minutes to inactivate the enzyme. Additionally, the purified products (0.5 μl) were mixed with 9 μl of Hi-Di formamide and 0.5 μl of the Lizl20 Size Standard (Applied Biosystems, USA) and separated in the ABI Prism 3730XL Genetic Analyzer (Applied Biosystems, USA). The final data were analyzed with GeneMapper 4.1 (Applied Biosystems, USA).

Freshly collected pancreatic tissues were frozen in liquid nitrogen and grounded into powder using mortar. Genome DNA of the pancreas was extracted using Purelink Kit (Invitrogen) according to the manufacturer’s protocol. Bisulfite treatment was performed using EZ DNA Methylation-Gold Kit (Zymo Research). The target sequence was cloned by Ex Taq HS (Takara Bio) and ligated into pCR4-Topo vector using Topo cloning technology (Invitrogen). These plasmids were transformed into DH5α at 37 °C, overnight. Colony PCR was performed using Go taq Master Mix (Promega) and ascertained ligation of the cassettes. After cleanup of the PCR products by exonuclease I (New England Biolabs) and shrimp alkaline phosphatase (Takara Bio), the sequence reaction was performed using M13 Rv primer (5′-CAGGAAACAGCTATGAC-3′) and was sequenced with ABI 3500 xL (Applied Biosystems). Methylation rates were measured using Quantification tool for Methylation Analysis (QUMA) software (Riken).