Regulation of 25- and 27-hydroxylation side chain cleavage pathways for cholic acid biosynthesis in humans, rabbits, and mice. Assay of enzyme activities by high-resolution gas chromatography;-mass spectrometry

In classic cholic acid biosynthesis, a series of ring modifications of cholesterol precede side chain cleavage and yield 5beta-cholestane-3alpha, 7alpha, 12alpha-triol. Side chain reactions of the triol then proceed either by the mitochondrial 27-hydroxylation pathway or by the microsomal 25-hydroxylation pathway. We have developed specific and precise assay methods to measure the activities of key enzymes in both pathways, 5beta-cholestane-3alpha, 7alpha, 12alpha-triol 25- and 27-hydroxylases and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases. The extracts from either the mitochondrial or microsomal incubation mixtures were purified by means of a disposable silica cartridge column, derivatized into trimethylsilyl ethers, and quantified by gas chromatography;-mass spectrometry with selected-ion monitoring in a high resolution mode. Compared with the addition of substrates in acetone, those in 2-hydroxypropyl-beta-cyclodextrin increased mitochondrial triol 27-hydroxylase activity 132% but decreased activities of the enzymes in microsomal 25-hydroxylation pathway (triol 25-hydroxylase and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases) 13;-60% in human liver. The enzyme activities in both pathways were generally 2- to 4-times higher in mouse and rabbit livers compared with human liver. In all species, microsomal triol 25-hydroxylase activities were 4- to 11-times larger than mitochondrial triol 27-hydroxylase activities but the activities of tetrol 24S-hydroxylase were similar to triol 27-hydroxylase activities in our assay conditions. The regulation of both pathways in rabbit liver was studied after bile acid synthesis was perturbed. Cholesterol feeding up-regulated enzyme activities involved in both 25- (64;-142%) and 27- (77%) hydroxylation pathways, while bile drainage up-regulated only the enzymes in the 25-hydroxylation pathway (178;-371%). Using these new assays, we demonstrated that the 25- and 27-hydroxylation pathways for cholic acid biosynthesis are more active in mouse and rabbit than human livers and are separately regulated in rabbit liver.     

What Happened before Losses of Photosynthesis in Cryptophyte Algae?

In many lineages of algae and land plants, photosynthesis was lost multiple times independently. Comparative analyses of photosynthetic and secondary nonphotosynthetic relatives have revealed the essential functions of plastids, beyond photosynthesis. However, evolutionary triggers and processes that drive the loss of photosynthesis remain unknown. Cryptophytes are microalgae with complex plastids derived from a red alga. They include several secondary nonphotosynthetic species with closely related photosynthetic taxa. In this study, we found that a cryptophyte, Cryptomonas borealis, is in a stage just prior to the loss of photosynthesis. Cryptomonas borealis was mixotrophic, possessed photosynthetic activity, and grew independent of light. The plastid genome of C. borealis had distinct features, including increases of group II introns with mobility, frequent genome rearrangements, incomplete loss of inverted repeats, and abundant small/medium/large-sized structural variants. These features provide insight into the evolutionary process leading to the loss of photosynthesis.     

Replication Study in a Japanese Population to Evaluate the Association between 10 SNP Loci,Identified in European Genome-Wide Association Studies,and Type 2 Diabetes

AimWe performed a replication study in a Japanese population to evaluate the association between type 2 diabetes and 7 susceptibility loci originally identified by European genome-wide association study (GWAS) in 2012: ZMIZ1, KLHDC5, TLE1, ANKRD55, CILP2, MC4R, and BCAR1. We also examined the association of 3 additional loci: CCND2 and GIPR, identified in sex-differentiated analyses, and LAMA1, which was shown to be associated with non-obese European type 2 diabetes.MethodsWe genotyped 6,972 Japanese participants (4,280 type 2 diabetes patients and 2,692 controls) for each of the 10 single nucleotide polymorphisms (SNPs): rs12571751 in ZMIZ1, rs10842994 near KLHDC5, rs2796441 near TLE1, rs459193 near ANKRD55, rs10401969 in CILP2, rs12970134 near MC4R, rs7202877 near BCAR1, rs11063069 near CCND2, rs8108269 near GIPR, and rs8090011 in LAMA1 using a multiplex polymerase chain reaction invader assay. The association of each SNP locus with the disease was evaluated using a logistic regression analysis.ResultsAll SNPs examined in this study had the same direction of effect (odds ratio > 1.0, p = 9.77 × 10^-4, binomial test), as in the original reports. Among them, rs12571751 in ZMIZ1 was significantly associated with type 2 diabetes [p = 0.0041, odds ratio = 1.123, 95% confidence interval 1.037–1.215, adjusted for sex, age and body mass index (BMI)], but we did not observe significant association of the remaining 9 SNP loci with type 2 diabetes in the present Japanese population (p ≥ 0.005). A genetic risk score, constructed from the sum of risk alleles for the 7 SNP loci identified by un-stratified analyses in the European GWAS meta-analysis were associated with type 2 diabetes in the present Japanese population (p = 2.3 × 10^-4, adjusted for sex, age and BMI).ConclusionsZMIZ1 locus has a significant effect on conferring susceptibility to type 2 diabetes also in the Japanese population.     

Metabolic Plasticity in Resting and Thrombin Activated Platelets

Platelet thrombus formation includes several integrated processes involving aggregation, secretion of granules, release of arachidonic acid and clot retraction, but it is not clear which metabolic fuels are required to support these events. We hypothesized that there is flexibility in the fuels that can be utilized to serve the energetic and metabolic needs for resting and thrombin-dependent platelet aggregation. Using platelets from healthy human donors, we found that there was a rapid thrombin-dependent increase in oxidative phosphorylation which required both glutamine and fatty acids but not glucose. Inhibition of fatty acid oxidation or glutamine utilization could be compensated for by increased glycolytic flux. No evidence for significant mitochondrial dysfunction was found, and ATP/ADP ratios were maintained following the addition of thrombin, indicating the presence of functional and active mitochondrial oxidative phosphorylation during the early stages of aggregation. Interestingly, inhibition of fatty acid oxidation and glutaminolysis alone or in combination is not sufficient to prevent platelet aggregation, due to compensation from glycolysis, whereas inhibitors of glycolysis inhibited aggregation approximately 50%. The combined effects of inhibitors of glycolysis and oxidative phosphorylation were synergistic in the inhibition of platelet aggregation. In summary, both glycolysis and oxidative phosphorylation contribute to platelet metabolism in the resting and activated state, with fatty acid oxidation and to a smaller extent glutaminolysis contributing to the increased energy demand.     

Metal ion dependency of serine racemase from Dictyostelium discoideum

D-Serine is known to act as an endogenous co-agonist of the N-methyl-D-aspartate receptor in the mammalian brain and is endogenously synthesized from L-serine by a pyridoxal 5'-phosphate-dependent enzyme, serine racemase. Though the soil-living mycetozoa Dictyostelium discoideum possesses no genes homologous to that of NMDA receptor, it contains genes encoding putative proteins relating to the D-serine metabolism, such as serine racemase, D-amino acid oxidase, and D-serine dehydratase. D. discoideum is an attractive target for the elucidation of the unknown functions of D-serine such as a role in cell development. As part of the elucidation of the role of D-serine in D. discoideum, we cloned, overexpressed, and examined the properties of the putative serine racemase exhibiting 46% amino acid sequence similarity with the human enzyme. The enzyme is unique in its stimulation by monovalent cations such as Na(+) in addition to Mg(2+) and Ca(2+), which are well-known activators for the mammalian serine racemase. Mg(2+) or Na(+) binding caused two- to ninefold enhancement of the rates of both racemization and dehydration. The half-maximal activation concentrations of Mg(2+) and Na(+) were determined to be 1.2?μM and 2.2?mM, respectively. In the L-serine dehydrase reaction, Mg(2+) and Na(+) enhanced the k (cat) value without changing the K (m) value. Alanine mutation of the residues E207 and D213, which correspond to the Mg(2+)-binding site of Schizosaccharomyces pombe serine racemase, abolished the Mg(2+)- and Na(+)-dependent stimulation. These results suggest that Mg(2+) and Na(+) share the common metal ion-binding site.     

Role of proline residues in conferring thermostability on aqualysin I

To understand the molecular basis of the thermostability of a thermophilic serine protease aqualysin I from Thermus aquaticus YT-1, we introduced mutations at Pro5, Pro7, Pro240 and Pro268, which are located on the surface loops of aqualysin I, by changing these amino acid residues into those found at the corresponding locations in VPR, a psychrophilic serine protease from Vibrio sp. PA-44. All mutants were expressed stably and exhibited essentially the same specific activity as wild-type aqualysin I at 40 degrees C. The P240N mutant protein had similar thermostability to wild-type aqualysin I, but P5N and P268T showed lower thermostability, with a half-life at 90 degrees C of 15 and 30 min, respectively, as compared to 45 min for the wild-type enzyme. The thermostability of P7I was decreased even more markedly, and the mutant protein was rapidly inactivated at 80 degrees C and even at 70 degrees C, with half-lives of 10 and 60 min, respectively. Differential scanning calorimetry analysis showed that the transition temperatures of wild-type enzyme, P5N, P7I, P240N and P268T were 93.99 degrees C, 83.45 degrees C, 75.66 degrees C, 91.78 degrees C and 86.49 degrees C, respectively. These results underscore the importance of the proline residues in the N- and C-terminal regions of aqualysin I in maintaining the integrity of the overall protein structure at elevated temperatures.     

Rapid sexing of bovine preimplantation embryos using loop-mediated isothermal amplification

Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method that amplifies a target sequence specifically under isothermal conditions. The product of LAMP is detected by the turbidity of the reaction mixture without electrophoresis. The objective of this study was to develop a rapid sexing method for bovine preimplantation embryos using LAMP. The first experiment was conducted to optimize the DNA extraction method for LAMP-based embryo sexing. The DNA of single blastomeres was extracted using three methods: heat, NaOH, and proteinase K-Tween 20 (PK-TW) treatments. Sexing was performed with two LAMP reactions, male-specific and male-female common reaction, after DNA extraction. The rates of correct determination of sex were 88.9-94.4%, with no difference among methods. The sensitivity and accuracy of LAMP-based embryo sexing were evaluated in the next experiment. The proportion of samples in which the sex was correctly determined was 75-100% for one to five biopsied cells. Lastly, in vivo-derived embryos were examined to verify the usefulness of LAMP-based embryo sexing, and some of these fresh, sexed embryos were transferred into recipient animals. The time needed for sexing was <1 h. The pregnancy rate was 57.4% and all calves born were of the predicted sex (12 male and 21 female). Therefore, LAMP-based embryo sexing accurately determined gender and is suitable for field application.     

Genotyping over 100,000 SNPs on a pair of oligonucleotide arrays

We present a genotyping method for simultaneously scoring 116,204 SNPs using oligonucleotide arrays. At call rates >99%, reproducibility is >99.97% and accuracy, as measured by inheritance in trios and concordance with the HapMap Project, is >99.7%. Average intermarker distance is 23.6 kb, and 92% of the genome is within 100 kb of a SNP marker. Average heterozygosity is 0.30, with 105,511 SNPs having minor allele frequencies >5%.     

Synthesis of p-nitrophenyl sulfated disaccharides with beta-D-(6-sulfo)-GlcNAc units using beta-N-acetylhexosaminidase from Aspergillus oryzae in a transglycosylation reaction

When α-d-GlcNAc-OC₆H₄NO₂ -p and β-d-(6-sulfo)-GlcNAc-OC₆H₄NO₂-p (2) were used as substrates, β-N-acetylhexosaminidase from Aspergillus oryzae transferred the β-d-(6-sulfo)-GlcNAc(unit from 2 to α-d-GlcNAc-OC₆H₄NO₂ -p to afford β-d-(6-sulfo)-GlcNAc-(1→4)-α-d-GlcNAc-OC₆H₄NO₂-p (3) in a yield of 94% based on the amount of donor, 2, added. β-d-(6-sulfo)-GlcNAc-(1→4)-α-d-Glc-OC₆H₄NO₂-p (4) was obtained with α-d-Glc-OC₆H₄NO₂ -p as acceptor in a similar manner. With a reaction mixture of 2 and β-d-GlcNAc-OC₆H₄NO₂-p (1) in a molar ratio of 6:1, the enzyme mediated the transfer of β-d-GlcNAc from 1 to 2, affording disaccharide β-d-GlcNAc-(1→4)-β-(6-sulfo)-d-GlcNAc-OC₆H₄NO₂-p (5) in a yield of 13% based on the amount of 1 added.     

Label-free quantitative proteomics using large peptide data sets generated by nanoflow liquid chromatography and mass spectrometry

We developed an integrated platform consisting of machinery and software modules that can apply vast amounts of data generated by nanoflow LC-MS to differential protein expression analyses. Unlabeled protein samples were completely digested with modified trypsin and separated by low speed (200 nl/min) one-dimensional HPLC. Mass spectra were obtained every 1 s by using the survey mode of a hybrid Q-TOF mass spectrometer and displayed in a two-dimensional plane with m/z values along the x axis, and retention time was displayed along the y axis. The time jitter of nano-LC was adjusted using newly developed software based on a dynamic programming algorithm. The comprehensiveness (60,000-160,000 peaks above the predetermined threshold detectable in 60-microg cell protein samples), reproducibility (average coefficient of variance of 0.35-0.39 and correlation coefficient of over 0.92 between duplicates), and accurate quantification with a wide dynamic range (over 10(3)) of our platform warrant its application to various types of experimental and translational proteomics.