The quantitative level of PRDM1α mRNA was normalized to β-actin using the cycle threshold (Ct) method (2-△△Ct method). For each sample, 3 independent experiments were made with triplicates for each experiment. Samples from plasma cell myeloma and tonsil were used as positive controls for PRDM1α Blasticidin S datasheet mRNA detection. ISH detection ISH for miR-223, miR-886-3p, and miR-34c-5p was performed for 31 EN-NK/T-NTs, 10 peripheral T-cell lymphomas, and 13 inflammatory nasal mucosa specimens. The presence of NK cells within the inflammatory nasal mucosa specimens were identified

by CD56 selleck chemical immunostaining. Probes labelled with a locked nuclear acid (LNA)™ probe for miR-223, miR-886-3p, and miR-34c-5p were designed and generated by Bio Perfectus Technologies (Jiang-su, China) according to sequences in the miRbase (Table 1). CX-6258 supplier Table 1 Sequences of in situ hybridisation probes for miR-223, miR-886-3p,

and miR-34c-5p miRNA MiRbase no. Genomic location Probe hsa-miR-223 MIMAT0000280 Xq12 5′-TGGGGTATTTGACAAACTGACA-3′ hsa-miR-886-3p MIMAT0004906 5q31.1 5′-AAGGGTCAGTAAGCACCCGCG-3′ hsa-miR-34c-5p MIMAT0000686 11q23.1 5′-GCAATCAGCTAACTACACTGCCT-3′ The ISH assays for miRNAs were performed as follows: FFPE tissues were routinely deparaffinised in xylene and rehydrated with an ethanol gradient, treated with 1 mg/ml Proteinase K for 10 min at 37°C, fixed with 4% formaldehyde for 10 min, and then dehydrated in ice-cold 90% ethanol. A 20-μL volume of hybridisation mixture consisting of 2 μL of the indicated LNA™ probe and 18 μL of a solution of 200 μg/mL salmon sperm DNA, 1 mg/mL dithiothreitol (DTT), 50% formamide, 2× Denhardt’s, 1 mg/mL

polyglucosan, and 2× saline-sodium citrate (2× SSC) was applied to each slide. The hybridisation reactions were performed overnight at 42°C in a humidified chamber. The sections were stringently rinsed 3 times for 15 min each in 2× SSC at 37°C, and endogenous peroxidases were blocked with 10% H2O2 for 20 min. After 2 washes in 1× PBS for 10 min, the slides were blocked with goat serum (1:100) for 30 min. The slides were then incubated with mouse anti-digoxin antibody for 20 h at 4°C. The slides were washed twice with 1× PBS, incubated with polymer auxiliary agent for 30 min, and washed with 1× PBS next for 10 min. Goat anti-mouse secondary antibody was added to the slides. After 2 washes with 1× PBS, DAB staining was performed. miR-223-, miR-886-3p-, or miR-34c-5p-positive EN-NK/T-NT tissue was used as a positive control for miR-223, miR-886-3p, or miR-34c-5p staining, respectively. For negative control samples, the hybridisation reactions were performed with a sense probe. Cytoplasmic staining was interpreted as miRNA expression, and positive expression was defined as staining of 10% or more of the cells in each tumour. The grading was semi-quantitatively estimated as follows: negative (0% to <10%), weak (10% to ≤50% positive cells), or strong (>50% to 100% positive cells).

Methods Subjects and amino acid treatment protocol This randomized, placebo-controlled, double-blind trial was conducted with 36 male college volunteers who did not have any musculoskeletal disorders and had not partaken in any regular resistance training prior to the study (Table 1). The study was carried out in accordance with the Declaration of Helsinki and was approved by the Human Subjects Committee of the University of Tsukuba. All subjects provided written informed consent. Table 1 Grouping conditions and characteristics of subjects   Supply condition Physiological characteristics before experiment Group BCAA Taurine Age (years) Height (cm) Body W.

(kg) Fat (%) Muscle W. (kg) MVC (Nm) CIR (mm) PLCB selleck chemicals llc Placebo-1 Placebo-2 22.2 ± 1.1 170.8 ± 1.9 67.5 ± 3.5 18.3 ± 1.9 51.9 ± 1.8 36.5 ± 3.0 257.4 ± 7.6 BA 3.2 g Placebo-2 22.9 ± 1.1 176.7 ± 3.6 73.5 ± 4.3 20.1 ± 1.7 55.3 ± 2.4

41.9 ± 3.8 267.7 ± 7.9 TAU Placebo-1 2.0 g 22.2 ± 0.8 173.7 ± 2.2 65.5 ± 2.7 17.3 ± 1.3 51.7 ± 1.7 39.1 ± 2.4 251.3 ± 7.4 COMB 3.2 g 2.0 g 23.1 ± 1.3 174.5 ± 1.8 61.5 ± 1.6 14.2 ± 1.0 50.0 ± 1.3 35.0 ± 2.6 243.4 ± 6.6 Footnote: Data are expressed as means ± S. E. Proteasome inhibitor Abbreviation: PLCB, double-placebo control Selleckchem FG 4592 group; BA, branched-chain amino acids and placebo-2 supplement group; TAU, taurine and placebo-1 supplement group; COMB, combined (taurine and BCAA) supplement group; Placebo-1 and 2, placebo of BCAA and taurine supplementation, respectively (3.2g or 2.0g starch

mainly), Body W., body weight; Muscle W., muscle weight; MVC, maximal voluntary strength of isometric contraction; CIR, upper arm circumference. Subjects were randomly and equally divided into the following four groups (n = 9 per group): double-placebo control supplementation (PLCB); BCAA and placebo-2 supplementation (BA); taurine and placebo-1 supplementation (TAU); and BCAA and taurine supplementation (COMB). Subjects were orally administered two sachets containing a combination of BCAA (or placebo-1) and taurine (or placebo-2) after every meal for two weeks prior to exercise (Table 1 and Figure 1). We chose this timeframe because previous studies showed a significant Aldol condensation increase in muscular taurine concentration following two weeks of taurine administration in rats [18, 20], but not after one week in humans [21]. Since the present study was designed as a double-blind trial, the duration of BCAA supplementation prior to exercise was matched to the two-week duration of taurine supplementation. All subjects were instructed to fill out a supplemental checklist after every meal. The BCAA and taurine sachets contained 3.2 g (9.6 g/day) of a BCAA mixture (Ile: Leu: Val = 1:2:1; Aminofeel®, Seikatsu Bunkasya Co. Inc., Chiba, Japan) and 2.0 g (6.0 g/day) of taurine, respectively.

0. SOD activity in erythrocytes was measured

according to Misra and Fridovich (1972) methods. The activity was determined at 37 °C by the absorbance increase at 480 nm. Activity of SOD was expressed in adrenaline units (U/g Hb/100 mL). Haemoglobin concentrations were carried out according to Van Kempen and Zijlstra (1961). Total antioxidant status determination Determination of the total antioxidant status in blood plasma was performed by spectrophotometric method according to procedure no. NX2332 by Randox (Randox Laboratories Ltd., United Kingdom,). In brief, ABTS (2,2′-azino-di-[3-ethylbenzthiazoline sulphonate]) was incubated with peroxide (metmyoglobin) and H2O2 to produce the radical cation ABTS Cilengitide with a relatively stable blue-green colour. Antioxidants when added in examined sample caused suppression of this colour production measured as decrease of absorbance with a spectrometer (UV/Vis Spectrometer Lambda 14P, Perkin Elmer, USA) at 600 nm. The total antioxidant status was calculated as concentration

of antioxidants (mM). The electrochemical properties The electrochemical properties of ligands and metal ion complexes have been studied by cyclic voltammetry in DMF solution. Voltammetric measurements were made with the aid PGSTAT12 AUTOLAB electrochemical analyzer. Three electrodes were utilized in this system, a glassy carbon working electrode (GCE), a platinum wire auxiliary electrode and silver wire in contact with 0.1 M AgNO3 in ACN reference

electrode. The GCE with 3.0-mm diameter was manually cleaned with 1 µm alumina polish prior each scan. All solutions were deareated for 10 min prior MDV3100 to measurements with pure argon and then a blanket atmosphere of argon was maintained over the solution during measurements. The potentials were measured in 0.2 M [nBu4N][BF4]/DMF as supporting electrolyte, using the [Fe(η5-(C5H5)2] in DMF (E 1/2 = +0.72 V) as internal standard. Cell viability Cell viability was determined after 44 h of culturing of A375 cells in the presence of tested compounds at indicated concentrations. An acid phosphatase activity (APA) assay was used to assess viable cell numbers in Dolutegravir ic50 cultures. In brief, the plates were centrifuged at the indicated time points, the medium was discarded and replaced with 100 μL assay buffer containing 0.1 M sodium acetate (pH 5), 0.1 % Triton X-100 and 5 mM p-nitrophenyl phosphate (pNPP; Sigma-Aldrich, St. Louis, MO) and incubated for additional 2 h at 37 °C. The reaction was stopped with 10 μL of 1 M NaOH, and the absorbance values were measured at the Capmatinib molecular weight wavelength of 405 nm using a microplate reader (Infinite M200Pro, Tecan, Austria). Measurement of intracellular ROS ROS levels were evaluated by flow cytometry using the probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA; Sigma-Aldrich, St. Louis, MO, USA) as described previously (Lesiak et al., 2010). In brief, A375 melanoma cells (a gift from Prof.

0) preheated to 80°C, maintaining this temperature and keeping sections in this solution for 10 min in a microwave pressure cooker. After allowing the sections to cool to room temperature, the slides were rinsed in PBS (pH 7.4). Endogenous peroxidase activity was blocked by incubation of the tissue samples for 10 min in 3% hydrogen peroxide. Samples were incubated for 45 min with the primary antibodies at room temperature in a moisture chamber. VEGF determination and analysis Samples were incubated with mouse anti-VEGF monoclonal antibody (1:100) find more (Abcam, Cambridge

MA, USA) in BSA 1% in PBS for 45 min. After washing with PBS, binding of the primary antibodies was revealed by incubation for 20 min with LSAB+ System Link (DAKO, Carpinteria, CA, USA) and LSAB+ HRP, (Streptavidin HRP kit, DAKO). The slides were rinsed with PBS and exposed to diaminobenzidine for 5 min. After washing with PBS and counter-staining with hematoxylin, the slides were dehydrated by graduated alcohols and xylol, and mounted with Poly-mount. Numerical proportions of stained cells were established by analyzing 10 high-power fields (400×) in each section.

Only cytoplasmic staining was considered positive. Intensity was graded on a semi-quantitative scale from 0–3. Graduation of expression was considered negative if fewer than 5% of cells were stained. Determination of vascular density The samples were incubated for 45 min with mouse anti-CD34 monoclonal antibody (1:200) 4-Aminobutyrate aminotransferase (Biocare Medical, Concord, CA, USA) as a marker for vascular endothelial cells. Three separated, highly vascularized areas (“”hot spots”"), GS-1101 previously identified in high-power fields (100×, then 400×), were analyzed by two pathologists by means of optic microscopy without previous knowledge of hCG determinations. Any immunostained vessel clearly separated from adjacent vessels with no muscular wall and within the optic field was considered a neovascularization vessel.

Vascular density (VD) was considered as the average of the three evaluated zones. Statistical analysis For descriptive purposes, continuous variables were summarized as arithmetic means, medians, and standard NSC 683864 mouse deviations (SDs), while categorical variables were expressed as proportions and confidence intervals (CIs). Inferential comparisons were carried out using the Student t or the Mann-Whitney U test, according to data distribution determined by the Kolmogorov-Smirnov test. Chi square or Fisher exact test was used to assess significance between categorical variables. Statistically significant and borderline-significant variables (p < 0.1) were included in the multivariate logistic regression analysis. Overall survival time was measured from day of surgery to date of death or last follow-up visit and analyzed with the Kaplan-Meier method, and comparisons among sub-groups were performed with the log-rank test. For survival curve analysis, all variables were dichotomized.

Figure 3 Muscle glycogen concentration see more following the 16 day dietary intervention and exercise trial day, which consisted of a resting (rest) muscle biopsy, another following 60 min cycling at 70% VO 2 max (70%) , time to fatigue at 90% VO 2 max (90%) and at the end of 6 h recovery (6 h recovery). Carbohydrate (CHO) and carbohydrate and whey protein

isolates (CHO + WPI) trial were similar at rest. All time points following exercise were lower than rest in both trials (# P < 0.05). CHO + WPI trial was increased selleckchem from 90% VO2 max to end of 6 h recovery (* P < 0.05). Values are means ± SEM (n = 6). Figure 4 Glycogen synthase mRNA expression for the carbohydrate (CHO) and carbohydrate and whey protein isolates (CHO + WPI) trials. No differences were observed. Values are means ± SEM (n = 6). AMPK-α2 mRNA expression (Figure 5) was similar for CHO and CHO + WPI trials. Following cycling at 90% VO2 max

and end of 6 h recovery, the CHO trial was lower compared to rest (P < 0.05). PGC-1α mRNA expression (Figure 6) was significantly higher at the end of 6 h recovery compared to all other time points in the CHO + WPI trial (P < 0.05). Following 6 h recovery the CHO + WPI trial was significantly higher (P < 0.05) compared to the isocaloric carbohydrate matched CHO trial. Figure 5 AMPK-α2 mRNA expression for carbohydrate (CHO) and carbohydrate and whey protein isolates (CHO + WPI) trials. CHO group is significantly different https://www.selleckchem.com/PARP.html from rest to 90% and rest to end recovery (* P < 0.05). Values are mean ± SEM (n = 6). Figure 6 PGC-1α mRNA expression for carbohydrate (CHO) and carbohydrate and whey protein isolate trials (CHO + WPI) following 16 day dietary intervention and exercise trial. Muscle biopsies were taken at rest, another following 60 min cycling at 70% VO2 max (70%), time to fatigue at 90% VO2 max (90%) and at the end of 6 h recovery (6 h recovery). CHO + WPI trial was significantly lower at rest, following cycling at 70% and 90% VO2  max, compared to 6 h recovery

aminophylline (# P < 0.05). After 6 h of recovery the CHO + WPI trial was significantly increased compared to CHO trial (^P < 0.05). Values are mean ± SEM (n = 6). Discussion Protein is considered a key nutritional component for athletic success, however there appears to be a lack of information regarding the effect of combined CHO and protein supplementation on exercise adaptations during recovery. This study compared 2 weeks co-ingestion of whey protein isolates supplementation combined with a high carbohydrate diet with an iso-caloric carbohydrate matched diet in endurance athletes. Protein supplementation with adequate carbohydrate availability, included in a regular training program, did not influence intense aerobic cycling performance or pre- and post-exercise muscle glycogen levels.

jejuni NCTC 11168 cj0596 mutant

was significantly deficient in its ability to adhere to host cells [29]. The discrepancy in adherence results seen between the previous study and our current work could be due to strain differences, however, we cannot exclude the possibility that the previously obtained adherence phenotype was due to an unlinked mutation in the uncomplemented NCTC 11168 cj0596 mutant. The increased motility and invasiveness could be due to an increase in chemotaxis, or to increased flagellar function because of a change in outer membrane architecture or cell morphology that provides a motility advantage. Several proteins located on the cell surface play a role in the initial cell-to-cell contact that is a component of intestinal colonization selleck chemicals by C. jejuni. Because Cj0596 is thought to be involved in selleck folding outer membrane proteins, its mutation is likely to have an effect on surface-exposed proteins, which could affect the ability to colonize

the host intestinal tract. When mice were inoculated individually with the wild-type, mutant, or revertant, the cj0596 mutant initially was able to colonize at mean levels comparable to the wild-type and revertant strains. However, the mutant became increasingly colonization CYT387 ic50 deficient over time. The differences were statistically significant at days 21 and 28, but not at day 35 due to increased clearance of the wild-type and revertant strains from some mice. This colonization defect is likely not the result of the increased motility of the mutant, since motility typically correlates with better Branched chain aminotransferase animal colonization. One possible explanation for the decreased colonization ability of the mutant is that Cj0596 is required for the proper presentation

of surface structures that are necessary for mouse colonization (e.g., known or unknown adhesins, oxidative stress, or other mouse colonization factors). Additionally, when the mutant was placed in direct competition with the wild-type, it demonstrated an inability to compete with the wild-type for colonization of the mice. In competition experiments, curiously, colonization levels of both the wild-type and mutant were significantly lower (compared to individual infections), suggesting some sort of interference of these strains with each other. The cj0596 mutant shows elevated autoaggregation and biofilm formation (manuscript submitted), so it is possible that these or other features impacting C. jejuni community structure could be involved. In an effort to determine some of the molecular causes of the altered virulence phenotypes discussed previously, we conducted a proteomic analysis comparing the whole-cell protein profiles of wild-type, mutant, and revertant. As expected, CAT was found only in the mutant and Cj0596 was absent in the mutant, confirming the replacement of cj0596 with the cat cassette in the mutant, and restoration of Cj0596 expression in the revertant.

In Figure 2, measurement point coordinate P and normal vector N are shown in Equations 1 and 2, in regard to coordinate system F. (1) (2) Figure 2 Overall coordinate system in this measurement. F, the coordinate system of the optical system. W, a coordinate system of the sample system. S, the coordinate system of the main body of sample. Because

there is the distance of coordinate system www.selleckchem.com/products/cb-839.html F and coordinate system W ‘L−Δy + R y ’ apart on Y 1-axis, in regard to coordinate system W, measurement point coordinate P is expressed by the coordinate transformation that Equation 1 is translated. In regard to coordinate system W, normal vector N becomes the same as coordinate system F. Therefore, Equation 3 translated Equation 1, in regard to coordinate system W. (3) In regard to coordinate system S, when measurement point coordinate P and normal vector N are also translated, they become Equations 1 and 2, respectively. (4) (5) Here, the shape derived by using y and n y has low precision. Therefore, the shape is derived by

assigning P(x, z) and N(n x , n y ) to derivation algorithm. This profiler determines the surface shape from the normal vectors and their coordinates by rotational motion, which is more accurate than linear motion and requires no reference optics. Therefore, there are no limitations on the measured shape, and free-forms can be directly measured [11]. Algorithm for obtaining the surface profile We developed an algorithm for selleck compound calculating the three-dimensional surface profile from the acquired normal vectors and their coordinates. A normal vector is equivalent to the surface slope or derivative of the surface profile. In this algorithm, to derive a figure from a normal vector and the coordinate, we express the figure by a model function and then fit the differential calculus function (slope function) to data on the normal vector by using the least-squares method. By calculating each coefficient of the series, the surface profile is determined. very Equations 6 and 7 represent the surface shape and slope for the two-dimensional case, respectively;

the same approach applies to the three-dimensional case. (6) (7) (8) (f j , normal vector or slope; x j , its coordinates). High-speed nanoprofiler Figures 3 and 4 show a photograph and a schematic view, respectively, of the newly developed nanoprofiler for normal vector tracing. The maximum mass of the main body of this machine is approximately 1,200 kg. The measurement sample can set up a greatest dimension to Φ = 50 mm × 40 mm, with a maximum mass of 1 kg and an optical pass length of 400 mm between the sample and the detector. Additionally, each optical element is set by the alignment that a laser beam changes 10 nm on QPD, when a normal vector changes 0.1 μrad. This machine has two pairs of two-axis rotational stages with resolutions of 0.17 μrad and one linear motion stage with a resolution of 1 nm.

The DNA microarray profile of ST30-IVc [2B]/t019 is homogeneous with the South Western Pacific (SWP) ST30-IV clone as is therefore not considered a WA CA-MRSA. WA68 harbors a type D IEC and tst-1genes. www.selleckchem.com/products/CP-673451.html Clonal Complex 45 CC45 contains four PVL negative strains. Based on the agr group/capsule type the four isolates are divided into two groups which are further divided into subgroups based on the SCCmec

type. Group 1 agr group I/capsule 8 (two strains) i. SCCmec IVa [2B] contains WA75 (ST45/t1424). ii. SCCmec V [5C2] contains WA4 (ST45/t123) which harbors tst1 genes. Both strains harbor a type B IEC. The spa types are not closely related. Group 2 agr group IV/capsule type 8 (two strains) i. SCCmec IVc [2B] contains WA23 (ST45/t1575) ii. SCCmec V [5C2&5] contains WA84 (ST45/t1081). Both strains harbor a type B IEC and closely related spa types. Clonal Complex 59 CC59 agr type I/capsule

type 8 contains seven strains. The DNA microarray profiles of ST59/ST952-V [5C2&5] t437/t1950 are homogeneous with the Taiwan clone and therefore are not considered WA CA-MRSA [32]. Based on the SCCmec types the remaining five strains are divided into three subgroups: i. SCCmec Selleck SBE-��-CD IVa [2B] contains PVL positive WA55 and WA56 (ST59/t437). WA55 harbors a type B IEC while WA56 a type A IEC. ii. SCCmec IVb [2B] contains two PVL negative strains with unrelated spa types: WA73 (ST59/t528) and WA24 (ST87 [ST59slv]/t216). WA73 harbors a type C IEC (chp+scn) and WA24 a type B IEC. iii. SCCmec IVa [2B]&5 contains PVL negative WA15 (ST59/t976)

which harbors a type A IEC. Clonal Complex 72 CC72 contains two agr group I/capsule type 5 strains with closely related spa types. Based on the SCCmec type the two strains are divided into two subgroups: i. SCCmec IVa [2B] contains PVL positive WA44 (ST72/t791) harboring a type B IEC. ii. SCCmec V (5C2) contains PVL negative WA91 (ST72/t3092) harboring a type E IEC and tst1 genes. Clonal Complex 75 CC75 Vitamin B12 contains three PVL negative strains which are agr group/capsule nontypeable by DNA microarray: WA8 (ST75-IVa [2B]), WA79 (ST75-IVa [2B]) and WA72 (ST1304 [ST75slv]-IVa [2B]) [33]. The three strains have the same spa sequence (259-23-23-17-17-17-23-23-23-17-16) which has not been allocated a spa type number by the Ridom website. The three strains harbor a type E IEC. Clonal Complex 80 CC80 contains three PVL positive agr group III/capsule type 8 strains: ST80-IVc [2B]/t044, ST583 [ST80slv]-IVc [2B]/t044, and ST728 [ST80slv]-IVc [2B]/t044. The DNA microarray virulence profiles are identical with the European ST80-IV [2B] clone and therefore the three strains are not considered WA CA-MRSA. Clonal Complex 97 CC97 contains two PVL negative agr group I/capsule type 5 strains with closely related spa types: WA54 (ST953[ST97dlv]-IVa [2B]/t359) and WA63 (ST1174[ST97dlv]-IVa [2B]/t267). The strains harbor a type E IEC.

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The reactions were analysed with an ABI 310 (Applied Biosystems) or on an ABI 377 (Applied Biosystems) in which case Longranger Single Packs (Cambrex Bio Science, Rockland, Inc., Rockland, ME) were used. Sequence analysis Nucleotide sequences were analysed with computer programs based on those of Devereux et al. [16]. Sequence alignments were performed by using the Blast

programs [17] at the server of the National Center for Biotechnology Information, Bethesda, Md., USA http://​www.​ncbi.​nlm.​nih.​gov/​blast/​. Multiple sequence alignments and construction of the bootstrap tree were performed using ClustalX2.0 [18] Production of recombinant LadA Derivatives of the expression vector pQE32 containing wild type and mutated versions of ladA were transformed to E. coli M13 cells

(Qiagen). Transformation and purification of the recombinant proteins GDC 0449 using Ni-agarose (Qiagen) was performed according to the supplier’s instructions. Enzyme assays All enzyme assays were performed at 20°C. Dehydrogenase activities were determined using 100 mM glycine pH 9.6, 0.4 mM NAD+ and 100 mM substrate. Reductase activities were determined using 50 mM sodium phosphate pH 7.6, 0.2 mM NADH and 100 mM substrate. Absorbance changes at 340 nm (ε = 6.22 mM-1 cm-1) were measured on a Unicam UV-1 spectrophotometer (Spectronic Unicam, Rochester, NY). Sheep liver SDH was obtained from this website Sigma (S3764). Modelling Models of A. niger LadA and XdhA structures were generated using the SWISS-MODEL program http://​swissmodel.​expasy.​org/​/​SWISS-MODEL.​html[19–21] with a crystal structure of D-sorbitol dehydrogenase (Protein Data Bank code: 1PL6). In this structure human D-sorbitol dehydrogenase is in complex with the cofactor NAD and an inhibitor [12]. The models were represented using the software package PYMOL [22]. Site-directed mutagenesis Site directed

mutagenesis was performed using the Quik Change protocol (Stratagene, La Jolla, Calif.). Two complementary oligonucleotides of 30–34 nucleotides were designed for each mutation, carrying the mutation in the middle of the oligonucleotide. PCR mixtures contained 50 ng of DNA template, 125 ng of each oligonucleotide, 1 μl of a 10 mM dNTP stock, 5 μl of 10× pfu buffer, and sterile water to a total volume of 24 μl. Before the start of the PCR, 1 μl of Phospholipase D1 pfu DNA polymerase (Stratagene) was added. The reaction parameters were: denaturation of the DNA for 5 min at 95°C, followed by 16 cycles of 30 s denaturation (95°C), 1 min annealing (56°C) and 15 min amplification (68°C). The product was incubated for 4 h with DpnI at 37°C. This enzyme degrades methylated (template) DNA but not the DNA amplified during the PCR. Acknowledgements We would like to thank M. Pail and A. Wiebenga for technical assistance and J.M. van Aken for sequence analysis. LR was supported by the council for Chemical Sciences of the Netherlands Organization for Scientific Research (NWO-CW).