A new therapeutic strategy with istradefylline for postural deformities in Parkinson's disease.

AIM OF THE STUDY: Postural deformities are common in Parkinson's disease (PD) patients. Several treatment options have been reported, but responses to these treatments appear unpredictable. Istradefylline is a novel drug for PD. Cases of PD patients whose postural deformities were improved after withdrawal of dopamine agonists and initiation of istradefylline are presented. MATERIALS AND METHODS: Four consecutive patients with postural deformities including antecollis, Pisa syndrome, and camptocormia were recruited and treated with istradefylline in combination with withdrawal of dopamine agonists, which are possible causes of postural deformities. RESULTS: The dopamine agonists were discontinued an average of 26 months after the development of the postural deformities, and istradefylline was initiated an average of 1.3 months after dopamine agonist withdrawal. Three patients with preserved paraspinal muscle volume showed good responses to the treatment regimen at least two months after dopamine agonist withdrawal. CONCLUSIONS AND CLINICAL IMPLICATIONS: Postural deformities caused by dopamine agonists generally improve less than two weeks after dopamine agonist withdrawal. Given the response time in the present study, the response was unlikely to be caused solely by dopamine agonist withdrawal. Istradefylline can be a potential therapeutic option; however, appropriate selection of patients for treatment with istradefylline is warranted.

Cyclic adenosine monophosphate suppresses the transcription of proinflammatory cytokines via the phosphorylated c-Fos protein.

Intracellular cyclic adenosine monophosphate (cAMP) suppresses innate immunity by inhibiting proinflammatory cytokine production from monocytic cells. Enhanced expression of interleukin-10 (IL-10) has been suggested to be the mechanism of suppression. However, cAMP is still capable of suppressing production of the cytokines TNF-alpha and IL-12 in IL-10-deficient dendritic cells (DCs). Here, we demonstrated that the transcription factor c-Fos was responsible for the cAMP-mediated suppression of inflammatory cytokine production. c-Fos accumulated at high amounts in response to cAMP and lipopolysaccharide (LPS). Overexpression of c-Fos suppressed LPS-induced cytokine production, whereas cAMP-mediated suppression of TNF-alpha and IL-12 was impaired in Fos(-/-) DCs or in RAW264.7 cells treated with c-Fos siRNA. c-Fos physically interacted with p65 protein and reduced the recruitment of p65 to the Tnf promoter. Multiple sites of c-Fos were phosphorylated by the IKKbeta protein. Thus, we propose that c-Fos is a substrate of IKKbeta and is responsible for the immunosuppressive effect of cAMP.

Forced expression of stabilized c-Fos in dendritic cells reduces cytokine production and immune responses in vivo.

Intracellular cyclic adenosine monophosphate (cAMP) suppresses innate immunity by inhibiting proinflammatory cytokine production by monocytic cells. We have shown that the transcription factor c-Fos is responsible for cAMP-mediated suppression of inflammatory cytokine production, and that c-Fos protein is stabilized by IKKß-mediated phosphorylation. We found that S308 is one of the major phosphorylation sites, and that the S308D mutation prolongs c-Fos halflife. To investigate the role of stabilized c-Fos protein in dendritic cells (DCs) in vivo, we generated CD11c-promoter-deriven c-FosS308D transgenic mice. As expected, bone marrow-derived DCs (BMDCs) from these Tg mice produced smaller amounts of inflammatory cytokines, including TNF-α, IL-12, and IL-23, but higher levels of IL-10, in response to LPS, than those from wild-type (Wt) mice. When T cells were co-cultured with BMDCs from Tg mice, production of Th1 and Th17 cytokines was reduced, although T cell proliferation was not affected. Tg mice demonstrated more resistance to experimental autoimmune encephalomyelitis (EAE) than did Wt mice. These data suggest that c-Fos in DCs plays a suppressive role in certain innate and adaptive immune responses.

Whole cancer-region enhancement using meglumine-gadopentetate-glucose solution and 7.0-T magnetic resonance imaging.

To visualize whole cancerous region including hypoxic cancer without radiation exposure, we developed meglumine-gadopentetate-glucose solution for 7.0-T magnetic resonance imaging. The infusion solution consists of meglumine-gadopentetate and glucose solutions, and these solutions are mixed before the vein drip infusion. We used readily available solutions, and the concentrations of the meglumine-gadopentetate and glucose solutions were 37.14 and 5.0%, respectively. In the first and second experiments, vein infusions were conducted from a rabbit ear using meglumine-gadopentetate-saline and meglumine-gadopentetate-glucose solutions, and T1 weighted imaging was performed to visualize cancerous region. Using the meglumine-gadopentetate saline, it was not difficult to image cancer-growth regions with new blood vessels. Using the meglumine-gadopentetate-glucose solution, the signal intensity of whole cancerous region including hypoxic cancer substantially increased. The visualizing duration for the meglumine gadopentetate glucose was beyond 90 min, and the rabbit survived after the infusion. The signal intensity in the hypoxic cancer was increasing until 90 min using the meglumine-gadopentetate-glucose solution, since the meglumine-gadopentetate molecules were absorbed into almost the whole cancerous region along with glucose-molecule flows.

The modulation of CD40 ligand signaling by transmembrane CD28 splice variant in human T cells.

The role of CD40 ligand (CD40L)/CD40 signaling in T cell-dependent B cell differentiation and maturation has been amply documented. The mechanism of CD40 signaling in B cells has been well established, whereas the signaling mechanism of CD40L in T cell costimulation remains unknown. In this study we show that CD28i, a transmembrane splice variant of CD28 costimulatory receptor, complexes with CD40L in human T cells. The cross-linking of CD40L resulted in the coendocytosis of CD28i with CD40L. The tyrosine phosphorylation of CD28i followed the cross-linking of CD40L, and the overexpression of CD28i augmented the c-Jun NH2-terminal kinase, p21-activated kinase 2, and nuclear factor kappaB activation. These data indicate that CD28i, by functioning as a signaling adaptor, transduces CD40L signaling as well as CD28 signaling in human T cells.

Serum γ-glutamyltransferase, triglyceride and total cholesterol are possible prediabetic risk markers in young Japanese men.

Serum profiles of lipids and/or liver enzymes are established markers for the estimation of insulin resistance and diabetic risk in the non-diabetic middle-aged population. To identify prediabetic markers in young subjects, 110 young male subjects (20-29 years of age) with normal glucose tolerance (NGT) were divided into two groups by median body mass index (BMI), <22.18 (n=55) and ≥22.18 (n=55) kg/m(2). Indices of insulin sensitivity including HOMA-IR and ISI composite, indices of ß-cell function including HOMA-ß, insulinogenic index (ΔI(30)/ΔG(30)) and ΔI(30)/ΔG(30)/ HOMA-IR were calculated. Statistical associations between these parameters and the serum lipid profiles and liver function were evaluated. Alanine aminotransferase (ALT), γ-glutamyltransferase (GGT), total cholesterol (TC) and triglyceride (TG) levels were inversely correlated with the ISI composite among individuals with BMI ≥22.18 kg/m(2) but not those with BMI <22.18 kg/m(2). Multivariate regression analysis revealed that, in Group N, the plasma glucose levels at 60 min (PG(60)) were inversely correlated with the ISI composite and the insulinogenic index, and were positively correlated with the GGT, TC and TG levels. On the other hand, in Group L, PG(60) was correlated with the insulinogenic index, TC and TG levels. In conclusion, elevated levels of GGT, TC and TG are good clinical markers to predict diabetic risks, even in young NGT males. Of these, GGT was the most strongly related factor among subjects with relatively high BMI.

Peptidoglycan and lipopolysaccharide activate PLCgamma2, leading to enhanced cytokine production in macrophages and dendritic cells.

In macrophages and monocytes, microbial components trigger the production of pro-inflammatory cytokine through Toll-like receptors (TLRs). Although major TLR signaling pathways are mediated by serine/threonine kinases, including TAK1, IKK and MAP kinases, tyrosine phosphorylation of intracellular proteins by TLR ligands has been suggested in a number of reports. Here, we demonstrated that peptidoglycan (PGN) of a Gram-positive bacterial cell wall component, a TLR2 ligand and lipopoysaccharide (LPS) of a Gram-positive bacterial component, a TLR4 ligand induced tyrosine phosphorylation of phospholipase Cgamma-2 (PLCgamma2), leading to intracellular free Ca2+ mobilization in bone marrow-derived macrophages (BMMphi) and bone marrow-derived dendritic cells (BMDC). PGN- and LPS-induced Ca2+ mobilization was not observed in BMDC from PLCgamma2 knockout mice. Thus, PLCgamma2 is essential for TLR2 and TLR4-mediated Ca2+ flux. In PLCgamma2-knockdown cells, PGN-induced IkappaB-alpha phosphorylation and p38 activation were reduced. Moreover, PLCgamma2 was necessary for the full production of TNF-alpha and IL-6. These data indicate that the PLCgamma2 pathway plays an important role in bacterial ligands-induced activation of macrophages and dendritic cells.

Suppressor of cytokine signaling 1 protects mice against concanavalin A-induced hepatitis by inhibiting apoptosis.

UNLABELLED: Acute liver failure is associated with significant mortality. However, the underlying pathophysiological mechanism is not yet fully understood. Suppressor of cytokine signaling-1 (SOCS1), which is a negative-feedback molecule for cytokine signaling, has been shown to be rapidly induced during liver injury. Here, using liver-specific SOCS1-conditional-knockout mice, we demonstrated that SOCS1 deletion in hepatocytes enhanced concanavalin A (ConA)-induced hepatitis, which has been shown to be dependent on activated T and natural killer T (NKT) cells. Although serum cytokine level and NKT cell activation were similar in wild-type (WT) and SOCS1-deficient mice after ConA treatment, proapoptotic signals, including signal transducers and activators of transcription 1 (STAT1) and Jun-terminal kinase (JNK) activation, were enhanced in SOCS1-deficient livers compared with those in WT livers. SOCS1-deficient hepatocytes had higher expression of Fas antigen and were more sensitive to anti-Fas antibody-induced apoptosis than were WT hepatocytes. Furthermore, SOCS1-deficient hepatocytes were more sensitive to tumor necrosis factor (TNF)-alpha-induced JNK activation and apoptosis. These data indicate that SOCS1 is important to the prevention of hepatocyte apoptosis induced by Fas and TNF-alpha. In contrast, SOCS1 overexpression in the liver by adenoviral gene transfer prevented ConA-induced liver injury. CONCLUSION: These findings indicate that SOCS1 plays important negative roles in fulminant hepatitis and that forced expression of SOCS1 is therapeutic in preventing hepatitis.

HTLV-1 Tax-mediated TAK1 activation involves TAB2 adapter protein.

Human T cell leukemia virus type 1 (HTLV-1) Tax is an oncoprotein that plays a crucial role in the proliferation and transformation of HTLV-1-infected T lymphocytes. It has recently been reported that Tax activates a MAPKKK family, TAK1. However, the molecular mechanism of Tax-mediated TAK1 activation is not well understood. In this report, we investigated the role of TAK1-binding protein 2 (TAB2) in Tax-mediated TAK1 activation. We found that TAB2 physically interacts with Tax and augments Tax-induced NF-kappaB activity. Tax and TAB2 cooperatively activate TAK1 when they are coexpressed. Furthermore, TAK1 activation by Tax requires TAB2 binding as well as ubiquitination of Tax. We also found that the overexpression of TRAF2, 5, or 6 strongly induces Tax ubiquitination. These results suggest that TAB2 may be critically involved in Tax-mediated activation of TAK1 and that NF-kappaB-activating TRAF family proteins are potential cellular E3 ubiquitin ligases toward Tax.

Adaptor protein SH2-B linking receptor-tyrosine kinase and Akt promotes adipocyte differentiation by regulating peroxisome proliferator-activated receptor gamma messenger ribonucleic acid levels.

Adipocyte differentiation is regulated by insulin and IGF-I, which transmit signals by activating their receptor tyrosine kinase. SH2-B is an adaptor protein containing pleckstrin homology and Src homology 2 (SH2) domains that have been implicated in insulin and IGF-I receptor signaling. In this study, we found a strong link between SH2-B levels and adipogenesis. The fat mass and expression of adipogenic genes including peroxisome proliferator-activated receptor gamma (PPARgamma) were reduced in white adipose tissue of SH2-B-/- mice. Reduced adipocyte differentiation of SH2-B-deficient mouse embryonic fibroblasts (MEFs) was observed in response to insulin and dexamethasone, whereas retroviral SH2-B overexpression enhanced differentiation of 3T3-L1 preadipocytes to adipocytes. SH2-B overexpression enhanced mRNA level of PPARgamma in 3T3-L1 cells, whereas PPARgamma levels were reduced in SH2-B-deficient MEFs in response to insulin. SH2-B-mediated up-regulation of PPARgamma mRNA was blocked by a phosphatidylinositol 3-kinase inhibitor, but not by a MAPK kinase inhibitor. Insulin-induced Akt activation and the phosphorylation of forkhead transcription factor (FKHR/Foxo1), a negative regulator of PPARgamma transcription, were up-regulated by SH2-B overexpression, but reduced in SH2-B-deficient MEFs. These data indicate that SH2-B is a key regulator of adipogenesis both in vivo and in vitro by regulating the insulin/IGF-I receptor-Akt-Foxo1-PPARgamma pathway.

Beta-cell function is a major contributor to oral glucose disposition in obese Japanese students.

Determinants of glucose intolerance were studied in 163 obese Japanese young adults, 18 to 21 years old (43 females,120 males), who underwent 75-g oral glucose tolerance testing. Type 2 diabetes was newly diagnosed in 2.9% (n = 4); impaired fasting glucose (IFG) in 5.1% (n = 7); and impaired glucose tolerance (IGT) in 10.9% (n = 15). A homeostasis model assessment of insulin resistance (HOMA-IR) was used to estimate insulin sensitivity; beta-cell function during the first 30 min of the test was measured and defined as the insulinogenic index. This index was adjusted for insulin sensitivity, since this affects both beta-cell function and glucose disposition (disposition index). The relationship between insulinogenic index and 1/HOMA-IR was not hyperbolic. However, the disposition index (DI) was useful for the estimation of beta-cell function with the correct confirmation about it validity using beta-cell function index (BI). The association between insulin sensitivity and beta-cell function to glucose disposal, as measured by the area under the glucose curve (AUCg), was examined in all subjects. Insulin sensitivity was significantly related to AUCg (log HOMA-IR; R (2) = 0.142, p<0.0001). On the other hand, an inverse curvilinear relationship was observed between beta-cell function and AUCg (log(Delta I/Delta G)/HOMA-IR, R (2) = 0.411, p<0.0001). Thus, impaired beta-cell function, when estimated as DI, was strongly associated with impaired glucose disposal. In conclusion, our study showed that both insulin sensitivity and impaired beta-cell function are associated with impaired glucose metabolism, and that beta-cell function may be more important in determining glucose disposal.

[The effects of age on insulin sensitivity and insulin secretion in Japanese subjects with normal glucose tolerance].

The prevalence of impaired glucose tolerance and type 2 diabetes increases with age. However, controversial results have been reported in regard to which has a greater influence on the deterioration of glucose tolerance with age, namely impaired insulin sensitivity or impaired insulin secretion. The conflicting results may arise mainly from differences in the evaluation of insulin secretion and insulin sensitivity, and from differences in the physical composition and the ethnicity of the study subjects. We therefore selected Japanese subjects, between 20 and 80 years of age, with normal glucose tolerance (NGT) and with a body mass index (BMI) below 25.0 kg/m2, and then examined the subject's insulin sensitivity based on the indices of a homeostasis model assessment-insulin resistance index (HOMA-IR) and ISI composite (ISI), and beta-cell function by these of HOMA-beta, AUC I/G(0-120), an insulinogenic index (deltaI30/deltaG30), and then (deltaI30/deltaG30)/HOMA-IR derived from a 75 g-oral glucose tolerance test (OGTT). The subjects were divided into the six subgroups according to sex and age, below age 30, between ages 30 and 49, and equal to and over age 50. Both HOMA-IR and the ISI showed no differences across the range of age and sex. HOMA-beta decreased with age, and AUC I/G(0-120) decreased in the elderly. No change was observed in deltaI30/deltaG30 across the age range in men, however deltaI30/deltaG30/HOMA-IR, the index of the early phase insulin secretion adjusted for insulin sensitivity, decreased with age in both men and women. These data indicated that aging itself had no effect on insulin sensitivity, while insulin secretion in both the early and late phase during the OGTT deteriorated with age even within the NGT subjects.

Comprehensive evaluation of variability in nicotine metabolism and CYP2A6 polymorphic alleles in four ethnic populations.

Human cytochrome P450 (CYP) 2A6 metabolizes nicotine to cotinine and is a possible modulator of nicotine addiction. Quantitative and qualitative differences in nicotine addiction have been observed between ethnic groups. However, there are few data on the ethnic influences of the CYP2A6-nicotine metabolism relationship, particularly with regard to black subjects. We determined the nicotine metabolism and CYP2A6 genotype in 176 white subjects and 160 black subjects, comparing them with our previous data from 209 Korean subjects and 92 Japanese subjects. Large interindividual differences were observed in the cotinine/nicotine ratios in plasma calculated as an index of nicotine metabolism in white subjects (range, 0.6-36.5) and in black subjects (range, 0.9-30.4). No ethnic difference in the metabolic ratio was observed among white subjects (mean, 7.2 +/- 5.0), black subjects (mean, 7.1 +/- 4.7), and Korean subjects (mean, 8.7 +/- 11.9), whereas Japanese subjects showed a significantly (P < .005) lower metabolic ratio (mean, 3.8 +/- 3.1) compared with the other populations. Women showed significantly (P < .05) higher metabolic ratios than men in the black population (8.0 +/- 5.3 versus 6.0 +/- 3.7). Obvious ethnic differences in the CYP2A6 alleles were observed among these 4 populations. The combined frequencies of the alleles lacking or showing reduced enzymatic activity (CYP2A6*2, CYP2A6*4, CYP2A6*5, CYP2A6*7, CYP2A6*9, CYP2A6*10, CYP2A6*11, CYP2A6*17, CYP2A6*19, and CYP2A6*20) were 9.1%, 21.9%, 42.9%, and 50.5% in white, black, Korean, and Japanese subjects, respectively. These CYP2A6 alleles were associated with reduced nicotine metabolism. Among the homozygotes of CYP2A6*1, interindividual and ethnic differences in the metabolic ratio were still observed. Thus some factors other than genetic ones might also contribute to the interindividual and ethnic differences. This comprehensive study of 4 populations extends our understanding of nicotine metabolism and the impact of genetic polymorphisms of the CYP2A6 gene.

Assessment of sensations experienced by subjects during MR imaging examination at 7T.

PURPOSE: We investigated sensations experienced by a large number of subjects during magnetic resonance (MR) imaging examinations using a 7-tesla scanner and slow table-feed speed. METHODS: After examinations at 7T, 504 of 508 consecutive subjects completed questionnaires using an 11-point scale to rate 14 potential sensations and symptoms during table movement and stationary positioning of the table. We compared scores among the sensations and between table conditions and the mean values of the scores with those reported in previous studies and examined correlations between the scores and subject characteristics. RESULTS: Vertigo and feelings of curving or leaning in the right or left direction during table movement were experienced frequently and markedly compared to other sensations and sensations experienced when the table was stationary (P < 0.01) and were correlated with subject age and examination time (P < 0.05). However, moderate to severe (scores of 5 to 10) vertigo and a curving/leaning feeling during table movement were noted in only 10.5% (vertigo) and 10.9% (curving/leaning) of subjects, and the mean vertigo score, 1.26, appeared to be substantially lower than that reported in a previous study. Reports of a metallic taste, nausea, and light flashes were significantly rarer and weaker than other sensations (P < 0.05). CONCLUSION: Vertigo and feelings of curving during table movement were the most frequent sensations reported during MR imaging examination at 7T. However, the occurrence and severity were low and mild, presumably because of the slow table-feed speed, which suggests that most patients and volunteers found discomfort at 7T acceptable.

Effects of polymorphism in promoter region of human CYP2A6 gene (CYP2A6*9) on expression level of messenger ribonucleic acid and enzymatic activity in vivo and in vitro.

Cytochrome P450 (CYP) 2A6 catalyzes nicotine C-oxidation, leading to cotinine formation, a major metabolic pathway of nicotine in humans. There are genetic polymorphisms in the human CYP2A6 gene. Previously, we demonstrated that in vivo nicotine metabolism is impaired with the CYP2A6*4, CYP2A6*7, and CYP2A6*10 alleles in Japanese subjects and Korean subjects. An allele possessing a point mutation in the TATA box termed CYP2A6*9 (T-48G) has been reported to decrease the transcriptional activity in vitro as assessed by luciferase assay. In this study we investigated the effects of the CYP2A6*9 allele on in vivo enzymatic activity by evaluating nicotine metabolism. The mutation of T-48G was found only on the CYP2A6*1A allele but not on the CYP2A6*1B allele. Allele frequencies of CYP2A6*9 in Japanese subjects (n = 92) and Korean subjects (n = 209) were 21.3% and 22.3%, respectively. In Korean subjects the cotinine/nicotine ratios as an index of nicotine metabolism in the subjects with CYP2A6*9/CYP2A6*9 (4.3 +/- 2.4) were significantly lower than those in the subjects with CYP2A6*1A/CYP2A6*9 (7.7 +/- 5.6) and CYP2A6*1A/CYP2A6*1A (10.4 +/- 9.2) (P <.05 and P <.005, respectively). In Japanese subjects a similar result was observed, although it was not significant. Thus it is suggested that the mutation in the TATA box (CYP2A6*9 allele) caused the decreased in vivo enzymatic activity. With an in vitro study, it was shown that the expression levels of CYP2A6 messenger ribonucleic acid and coumarin 7-hydroxylase activity in human livers genotyped as CYP2A6*1/CYP2A6*9 and CYP2A6*9/CYP2A6*9 tended to be lower than those in human livers genotyped as CYP2A6*1/CYP2A6*1, although there was no significant difference because of the small number of samples. These in vitro data supported the in vivo data demonstrating that the CYP2A6*9 allele caused the decreased expression level and enzymatic activity of CYP2A6.

A novel polymorphism of human CYP2A6 gene CYP2A6*17 has an amino acid substitution (V365M) that decreases enzymatic activity in vitro and in vivo.

Cytochrome P450 (CYP) 2A6 is a major CYP responsible for the metabolism of nicotine and coumarin in humans. We identified a novel allele, designated CYP2A6*17 , which contains A51G (exon 1), C209T (intron 1), G1779A (exon 3), C4489T (intron 6), G5065A (V365M, exon 7), G5163A (intron 7), C5717T (exon 8), and A5825G (intron 8). We developed a genotyping method by polymerase chain reaction-restriction fragment length polymorphism for the CYP2A6*17 allele, targeting the G5065A mutation. The allele frequency in black subjects (n = 96) was 9.4% (95% confidence interval [CI], 5.3%-13.5%). The allele was not found in white subjects (95% CI, 0%-0.9%; n = 163), Japanese subjects (95% CI, 0%-1.6%; n = 92), and Korean subjects (95% CI, 0%-0.7%; n = 209). To examine the effects of the amino acid change in the CYP2A6*17 allele on the enzymatic activity, we expressed a wild-type or variant (V365M) CYP2A6 together with NADPH-CYP reductase in Escherichia coli . For coumarin 7-hydroxylation, the apparent Michaelis-Menten constant value of variant CYP2A6 (1.06 +/- 0.11 micromol/L) was significantly (P < .005) higher than that of wild type (0.60 +/- 0.05 micromol/L). The maximum velocity values of the wild-type and variant CYP2A6 were 0.61 +/- 0.06 and 0.64 +/- 0.07 pmol . min -1 . pmol -1 CYP, respectively. For nicotine C -oxidation, the apparent Michaelis-Menten constant values of the wild-type or variant CYP2A6 were 31.6 +/- 2.9 micromol/L and 31.3 +/- 3.1 micromol/L, respectively. The maximum velocity value of variant CYP2A6 (0.72 +/- 0.21 pmol . min -1 . pmol -1 CYP) was significantly (P < .05) lower than that of the wild type (1.80 +/- 0.42 pmol . min -1 . pmol -1 CYP). Thus the intrinsic clearance values for coumarin 7-hydroxylation and nicotine C -oxidation by the variant were both significantly (P < .05) decreased to 40% to 60% compared with the wild type. Furthermore, cotinine/nicotine ratios after 1 piece of nicotine gum was chewed, used as an index of in vivo nicotine metabolism, were significantly (P < .05) decreased in heterozygotes of the CYP2A6*17 allele (5.4 +/- 2.7, n = 12) compared with homozygotes of the wild type (11.5 +/- 10.5, n = 37). A subject with CYP2A6*17 / CYP2A6*17 revealed the lowest cotinine/nicotine ratio (1.8). We found a novel allele in black subjects that affects the nicotine metabolism in vitro and in vivo.

Novel human CYP2A6 alleles confound gene deletion analysis.

Cytochrome P450 (CYP) 2A6 metabolizes a number of drugs and a variety of procarcinogens. CYP2A6 also catalyzes nicotine C-oxidation leading to cotinine formation, a major metabolic pathway of nicotine in humans. There are genetic polymorphisms in the human CYP2A6 gene and a relationship between the CYP2A6 genotype and smoking habits as well as the incidence of lung cancer has been indicated. CYP2A6*4 alleles are the whole deleted type and are completely deficient in the enzymatic activity. An unequal crossover junction is located in the 3'-flanking region in the CYP2A6*4A allele, whereas the junction is located in either intron 8 or exon 9 in the CYP2A6*4D allele. In the present study, a novel genotyping method to distinguish between two different whole deleted alleles of CYP2A6*4A and CYP2A6*4D was established. In the process, two novel alleles, CYP2A6*1F and CYP2A6*1G, were found. The CYP2A6*1F has a single nucleotide polymorphism (SNP) of C5717T in exon 8, and the CYP2A6*1G has two SNPs, C5717T in exon 8 and A5825G in intron 8. The SNP of C5717T corresponds to C1224T on the cDNA sequence and is a synonymous mutation. Since the CYP2A6*1F produces a recognition site of the restriction enzymes that is the same as CYP2A6*4D, the presence of the CYP2A6*1F allele could cause a mistyping as the CYP2A6*4D allele. According to an improved genotyping method, the allele frequencies of CYP2A6*4A, CYP2A6*4D, CYP2A6*1F, and CYP2A6*1G in 165 Caucasians were 3.0%, 0%, 1.8%, and 1.2%, respectively. The allele frequencies of CYP2A6*4A, CYP2A6*4D, CYP2A6*1F, and CYP2A6*1G in 94 African-Americans were 0%, 0.5%, 0%, and 13.3%, respectively. This is the first report of a method that can distinguish between CYP2A6*4A, CYP2A6*4D, and CYP2A6*1F which could otherwise cause a mistyping as CYP2A6*4D.

Metabolic profile of nicotine in subjects whose CYP2A6 gene is deleted.

Generally, 70-80% of absorbed nicotine is mainly metabolized to cotinine by cytochrome P450 (CYP) 2A6. There is genetic polymorphism in the human CYP2A6 gene. Among several mutated alleles, CYP2A6*4 allele is a whole deleted type. The purpose of the present study was to clarify the metabolic profile of nicotine in subjects whose CYP2A6 gene is deleted. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for nicotine and its nine metabolites. Excretion levels of nicotine and its metabolites in 24 h accumulated urine after the chewing of one piece of nicotine gum were evaluated in five Japanese subjects whose CYP2A6 genotype was determined. In three subjects with CYP2A6*1A/CYP2A6*1A, CYP2A6*1A/CYP2A6*1B, and CYP2A6*1A/CYP2A6*4 (group I), nicotine was mainly excreted as cotinine, trans-3'-hydroxycotinine, and their glucuronide (approximately 60%). In contrast, in two subjects with CYP2A6*4/CYP2A6*4 (group II), trace levels of cotinine, cotinine N-glucuronide, and cotinine 1'-N-oxide were detected. Trans-3'-hydroxycotinine and its O-glucuronide were not detected. The excretion levels of nicotine itself, nicotine N-glucuronide, and nicotine 1'-N-oxide were higher than those in the other three subjects. The total excretion levels of these three compounds were approximately 95% in group II versus 35% in group I. However, the sum of the excretion levels of nicotine and all metabolites was similar among these five subjects. This is the first report of the metabolic profile of nicotine in subjects whose CYP2A6 gene is deleted.

Trinucleotide duplex formation inside a confined nanospace under supercooled conditions.

Self-assembly of nucleotides of fewer than three base pairs is often found in protein-nucleotide conjugations, despite their energetic instability, and is regarded as the potential starting point for the creation of artificial hydrogen-bonded supramolecular complexes. Here we report duplex formation of 3-mer DNA fragments confined within silica mesopores modified with a positively charged trimethyl aminopropyl monolayer, and their further stabilization under supercooled conditions (T<273 K). We load 3-mer DNA fragments with donor- or acceptor-dye into modified silica mesopores and examine their hybridization behaviours using FRET measurements. The FRET results clearly reveal that efficient duplex formation through at least two A-T base pairs can be achieved at 233 K. Enthalpy changes for duplex formation are found to be nearly equal between complementary and single-mismatched 3-mer DNA duplexes. These results confirm confined mesoscale cavities to be a novel low-temperature reaction space for hydrogen-bonded supramolecular complexes.

A novel mutation of CLCNKB in a Japanese patient of Gitelman-like phenotype with diuretic insensitivity to thiazide administration.

The clinical phenotypes of patients with Bartter syndrome type III sometimes closely resemble those of Gitelman syndrome. We report a patient with mild, adult-onset symptoms, such as muscular weakness and fatigue, who showed hypokalemic metabolic alkalosis, elevated renin-aldosterone levels with normal blood pressure, hypocalciuria and hypomagnesemia. She was also suffering from chondrocalcinosis. A diuretic test with furosemide and thiazide showed a good response to furosemide, but little response to thiazide. Although the clinical findings and diuretic tests predicted that the patient had Gitelman syndrome, genetic analysis found no mutation in SLC12A3. However, a novel missense mutation, p.L647F in CLCNKB, which is located in the CBS domain at the C-terminus of ClC-Kb, was discovered. Therefore, gene analyses of CLCNKB and SLC12A3 might be necessary to elucidate the precise etiology of the salt-losing tubulopathies regardless of the results of diuretic tests.