An enzyme combination assay for serum sphingomyelin: Improved specificity through avoiding the interference with lysophosphatidylcholine

Serum sphingomyelin (SM) has predictive value in the development of atherosclerosis. Furthermore, SM plays important roles in cell membrane structure, signal transduction pathways, and lipid raft formation. A convenient enzymatic method for SM is available for routine laboratory practice, but the enzyme specificity is not sufficient because of nonspecific reactions with lysophosphatidylcholine (LPC). Based on the differential specificity of selected enzymes toward choline-containing phospholipids, a two-step assay for measuring SM was constructed and its performance was evaluated using sera from healthy individuals on a Hitachi 7170 autoanalyzer. Results from this assay were highly correlated with theoretical serum SM concentrations estimated by subtracting phosphatidylcholine (PC) and LPC concentrations from that of total phospholipids determined using previously established methods. There was a good correlation between the results of SM assayed by the proposed method and the existing enzymatic method in sera from healthy individuals. Moreover, the proposed method was superior to the existing method in preventing nonspecific reactions with LPC present in sera. The proposed method does not require any pretreatment, uses 2.5 μl of serum samples, and requires only 10 min on an autoanalyzer. This high-throughput method can measure serum SM with sufficient specificity for clinical purposes and is applicable in routine laboratory practice.     

Reappraisal of VAChT‐Cre: Preference in slow motor neurons innervating type I or IIa muscle fibers

VAChT‐Cre.Fast and VAChT‐Cre.Slow mice selectively express Cre recombinase in approximately one half of postnatal somatic motor neurons. The mouse lines have been used in various studies with selective genetic modifications in adult motor neurons. In the present study, we crossed VAChT‐Cre lines with a reporter line, CAG‐Syp/tdTomato, in which synaptophysin‐tdTomato fusion proteins are efficiently sorted to axon terminals, making it possible to label both cell bodies and axon terminals of motor neurons. In the mice, Syp/tdTomato fluorescence preferentially co‐localized with osteopontin, a recently discovered motor neuron marker for slow‐twitch fatigue‐resistant (S) and fast‐twitch fatigue‐resistant (FR) types. The fluorescence did not preferentially co‐localize with matrix metalloproteinase‐9, a marker for fast‐twitch fatigable (FF) motor neurons. In the neuromuscular junctions, Syp/tdTomato fluorescence was detected mainly in motor nerve terminals that innervate type I or IIa muscle fibers. These results suggest that the VAChT‐Cre lines are Cre‐drivers that have selectivity in S and FR motor neurons. In order to avoid confusion, we have changed the mouse line names from VAChT‐Cre.Fast and VAChT‐Cre.Slow to VAChT‐Cre.Early and VAChT‐Cre.Late, respectively. The mouse lines will be useful tools to study slow‐type motor neurons, in relation to physiology and pathology.     

Gliclazide inhibits proliferation but stimulates differentiation of white and brown adipocytes

Gliclazide, a second-generation sulfonylurea, has anti-oxidant properties as well as hypoglycemic activities. In the present study, we investigated whether gliclazide affected proliferation and/or differentiation of HW white and HB2 brown adipocyte cell lines. Gliclazide inhibited proliferation of HW and HB2 cells in the medium containing fetal calf serum or epidermal growth factor (EGF). Gliclazide inhibited phosphorylation of EGF receptor and of extracellular signal-regulated kinase (ERK) 1/2 stimulated by EGF. Gliclazide increased lipid accumulation and peroxisome proliferator-activated receptor gamma (PPARgamma) expression in the early stage of differentiation of adipocytes. A K(ATP) channel activator, diazoxide, did not inhibit the increase of lipid accumulation by gliclazide. Furthermore, gliclazide inhibited the DNA-binding activity of PPARgamma in mature adipocytes. On the other hand, glibenclamide, other sulfonylurea, did not show these effects. These results indicate gliclazide inhibits proliferation and stimulates differentiation of adipocytes via down-regulation of the EGFR signalling. Gliclazide may have preventive and therapeutic effects on obesity, as well as on type 2 diabetes.     

Thrombomodulin is a clock-controlled gene in vascular endothelial cells

Cardiovascular diseases are closely related to circadian rhythm, which is under the control of an internal biological clock mechanism. Although a biological clock exists not only in the hypothalamus but also in each peripheral tissue, the biological relevance of the peripheral clock remains to be elucidated. In this study we searched for clock-controlled genes in vascular endothelial cells using microarray technology. The expression of a total of 229 genes was up-regulated by CLOCK/BMAL2. Among the genes that we identified, we examined the thrombomodulin (TM) gene further, because TM is an integral membrane glycoprotein that is expressed primarily in vascular endothelial cells and plays a major role in the regulation of intravascular coagulation. TM mRNA and protein expression showed a clear circadian oscillation in the mouse lung and heart. Reporter analyses, gel shift assays, and chromatin immunoprecipitation analyses using the TM promoter revealed that a heterodimer of CLOCK and BMAL2 binds directly to the E-box of the TM promoter, resulting in TM promoter transactivation. Indeed, the oscillation of TM gene expression was abolished in clock mutant mice, suggesting that TM expression is regulated by the clock gene in vivo. Finally, the phase of circadian oscillation of TM mRNA expression was altered by temporal feeding restriction, suggesting TM gene expression is regulated by the peripheral clock system. In conclusion, these data suggest that the peripheral clock in vascular endothelial cells regulates TM gene expression and that the oscillation of TM expression may contribute to the circadian variation of cardiovascular events.     

Reversibility of n-3 fatty acid deficiency-induced alterations of learning behavior in the rat: level of n-6 fatty acids as another critical factor

Rats fed a semipurified diet supplemented with 3% (w/w) safflower oil [Saf, n-3 fatty acid deficient, high linoleic acid (18:2n-6)] through two generations exhibit decreased correct response ratios in a brightness-discrimination learning test compared with rats fed 3% perilla oil [Per, high alpha-linolenic acid (18:3n-3)]. This is associated with a decreased DHA (22:6n-3)-to-arachidonic acid (20:4n-6) ratio in brain lipids. In the first set of experiments, dietary oil was shifted from Saf to a mixture of 2.4% safflower oil plus 0.6% DHA after weaning (Saf-DHA), but all parameters measured in the learning test were essentially unchanged. Brain 22:6n-3 content of the Saf-DHA group reached that of the Per group but the levels of 20:4n-6 and docosatetraenoic acid (22:4n-6) did not decrease to those of the Per group at the start of the test. In the second set of experiments, dietary oil was shifted to a mixture of 0.6% safflower oil plus 1.2% oleic acid (OA) plus 1.2% DHA (Saf-OA-DHA group) with 18:2n-6 content comparable to that of the Per group. The Saf-OA-DHA group exhibited a learning performance similar to that of the Per group; brain 22:6n-3, 20:4n-6, and 22:4n-6 contents were also comparable to those of the Per group. These results indicate that the altered learning behavior associated with a long-term n-3 fatty acid deficiency is reversed by supplementing 22:6n-3 after weaning, when the levels of competing n-6 fatty acids in the diet and brain lipids are limited.     

Metabolic engineering of the terpenoid biosynthetic pathway of Escherichia coli for production of the carotenoids β-carotene and zeaxanthin

Metabolic engineering of the early non-mevalonate terpenoid pathway of Escherichia coli was carried out to increase the supply of prenyl pyrophosphates as precursor for carotenoid production. Transformation with the genes dxs for over-expression of 1-deoxy-d-xylulose 5-phosphate synthase, dxr for 1-deoxy-d-xylulose 5-phosphate reductoisomerase and idi encoding an isopentenyl pyrophosphate stimulated carotenogenesis up to 3.5-fold. Co-transformation of idi with either dxs or dxr had an additive effect on ß-carotene and zeaxanthin production which reached 1.6 mg g^–1 dry wt.     

Imprinted polymer layer for recognizing double-stranded DNA

A method of preparing a thin polymer layer able to recognize double-stranded DNA (dsDNA) was developed by using 2-vinyl-4,6-diamino-1,3,5-triazine (VDAT) as a functional monomer for creating a DNA-imprinted polymer. The formation of hydrogen bonds between VDAT and A-T base pairs in dsDNA was confirmed by measuring the effects of VDAT on the melting point and the NMR and CD spectra of dsDNA. An imprinted polymer that can recognize dsDNA of the verotoxin gene was prepared by polymerizing VDAT, acrylamide, a crosslinking agent, and the template verotoxin dsDNA on a silanized glass surface. The specificity of this polymer layer for binding verotoxin dsDNA was investigated by using fluorescent-labelled dsDNAs. The fluorescence intensity of the polymer layer after binding verotoxin dsDNA was twice as high as after binding oligo(dG)-oligo(dC), indicating that verotoxin dsDNA was preferentially bound to the polymer imprinted with verotoxin dsDNA. The kinetics of verotoxin dsDNA binding to the imprinted polymer were analyzed by surface plasmon resonance measurements. The dissociation constant (KD) was low, of the order of 10(-9)M.     

Synthesis and evaluation of 6-methylene-bridged uracil derivatives. Part 2: optimization of inhibitors of human thymidine phosphorylase and their selectivity with uridine phosphorylase

A series of novel 6-methylene-bridged uracil derivatives have been optimized for clinical use as the inhibitors of human thymidine phosphorylase (TP). We describe their synthesis and evaluation. Introduction of a guanidino or an amidino group enhanced the in vitro inhibitory activity of TP comparing with formerly reported inhibitor 1. Their selectivity for TP based on uridine phosphorylase inhibitory activity was also evaluated. Compound 2 (TPI) has been selected for clinical evaluation based on its strong TP inhibition and excellent modulation of 2'-deoxy-5-(trifluoromethyl)uridine (F(3)dThd) pharmacokinetics. As a result, TAS-102 (a combination of F(3)dThd and TPI) is currently in phase 1 clinical studies.     

A series of immune responses leading to the induction ofT cell IL-12/IL-18 responsiveness in patientswith relatively large tumor burdens

The induction of interleukin-12 (IL-12) responsiveness in T cells depends on T cell receptor (TCR) triggering, and is regarded as a parameter of recently TCR-sensitized T cells. Here, we investigated whether IL-12 responsiveness could be detected in freshly prepared T cells from tumor-bearing patients, and if so whether such patients exhibited additional immunological parameters related to IL-12 responsiveness. CD4(+) and CD8(+) T cell populations from an appreciable proportion of tumor-bearing patients exhibited high levels of IL-12 responsiveness as evaluated by IL-12-stimulated interferon-gamma (IFN-gamma) production. T cell populations with high IL-12 responsiveness were observed in the group of patients with moderate to large tumor mass or tumor metastases rather than in patients with small tumors. The frequency of such a T cell population was also lower in post-surgery tumor-free patients, showing the correlation between IL-12 responsiveness and the presence of a certain extent of tumor burden. More importantly, a higher incidence of IL-12 responsiveness was observed in tumor-bearing patients exhibiting detectable plasma IL-12 levels, and correlated with IL-18 responsiveness. T cell IL-12 and IL-18 responsiveness is induced by TCR triggering and subsequent IL-12 stimulation respectively. Furthermore, TCR-triggered T cells stimulate antigen-presenting cells (APC) to produce IL-12. Therefore, the present observations suggest that an immune response loop from TCR sensitization to the induction of IL-12/IL-18 responsiveness via IL-12 production operates in tumor-bearing patients, particularly in those with relatively large tumor burdens.     

Effect of group selection on the evolution of altruistic behavior

By using a Monte Carlo simulation, we studied the effect of group selection on the altruistic trait that is controlled by a single locus. The altruistic trait is disadvantageous to the bearer but advantageous to the others. Group selection is defined as the differential reproductive rate among demes caused by genotypic difference among demes. We found that the simulation reproduced many results of former studies. Additionally, when the mutation rate and the migration rate are small enough, we observed two new phenomena: (1) When the effect of the group selection is as large as that of the individual selection, the gene frequency is quite unstable. We found two local stable states, the A- and the S-state. When the metapopulation is in the A-state, altruists are nearly fixed. When in the S-state, on the contrary, altruists are almost lost. The metapopulation shifted quickly from one state to another. We call this phenomenon as the S-A transition. (2) When the mutation rate and migration rate are small enough we found an extremely strong mechanism to stop the non-altruists from expanding no matter how strong the individual selection coefficient is. This is caused by a phenomenon, which we call SA splitting, in which most demes are fixed either by altruists or non-altruists; thus, the relatedness of the metapopulation becomes nearly equal to one. We show SA splitting plays an important role in S-A transition. We define a parameter d to see the degree of SA splitting. We found that d is roughly proportional to mutation rate and deme size.