A possible connection between tumor necrosis factor alpha and adropin levels in polycystic ovary syndrome.

CONTEXT: Adropin is a peptide hormone implicated in the regulation of insulin sensitivity and energy homeostasis. Polycystic ovary syndrome (PCOS) is a metabolic and reproductive disease associated with insulin resistance. It has been demonstrated that various inflammatory markers increased in PCOS including TNF-α. TNF-α regulates the secretion of certain peptides which play a crucial role in glucose and lipid homeostasis. There is also some evidence of a link between TNF-α and adropin. OBJECTIVE: To ascertain whether there is an association between circulating adropin levels and TNF-α in PCOS. PATIENTS AND DESIGN: 152 women with PCOS and 152 age- and body mass index-matched controls without PCOS were recruited for this cross-sectional study. MAIN OUTCOME MEASURES: Adropin and TNF-α levels were measured using ELISA. RESULTS: Adropin levels were lower in the PCOS group compared with the control group (7.43 ± 0.79 vs. 9.42 ± 0.76 ng/ml, P < 0.001), whereas TNF-α levels were higher (49.93 ± 3.39 vs. 35.83 ± 2.47 pg/ml, P < 0.001). A strongly negative correlation was found between circulating adropin levels and TNF-α levels in women with PCOS (r = -0.407, P < 0.001). Binary logistic regression analysis revealed that decreased adropin levels were significantly associated with high odds of having PCOS, although, after adjustment for TNF-α, this link vanished. Additionally, multiple linear regression analysis showed that HOMA-IR and TFN-α independently predicted adropin levels. CONCLUSIONS: Serum adropin levels are significantly decreased in PCOS and are inversely associated with TNF-α. Further dissection of the nature of this association can open new therapeutic options for metabolic diseases.

Serum nesfatin-1 and leptin levels in non-obese girls with premature thelarche.

AIM: We aimed to investigate serum nesfatin-1 level in girls with premature thelarche (PT) and its relationship with anthropometric parameters and leptin, which are involved in the initiation of pubertal process. SUBJECTS-METHODS: Non-obese girls who presented with the complaint of early (2-8 years) and isolated breast development were included in the study. The control group consisted of age-matched healthy prepubertal girls. Auxological measurements were performed in all subjects. Gonadotropin-releasing hormone (GnRH) stimulation test and bone age assessment were conducted in subjects with early breast development. Girls with a bone age/chronologic age ratio <1.2 and a peak luteinizing hormone (LH) response to GnRH stimulation <5 mIU/L were included in the PT group. RESULTS: The study included 22 non-obese girls with PT and 24 healthy prepubertal controls. Body mass index (BMI), BMI-standard deviation score (SDS) and height SDS were similar between the groups (p > 0.05). Serum leptin and nesfatin-1 levels were found significantly higher in the PT group compared to controls (p < 0.05). No correlation was detected between nesfatin-1 and basal LH, basal follicle stimulating hormone (FSH), stimulated peak LH, peak FSH, leptin levels and anthropometric parameters in the PT group (p > 0.05). CONCLUSION: Results of the present study showed that serum nesfatin-1 and leptin levels are significantly higher in girls with PT than in prepubertal controls. This finding suggests that similar to leptin, nesfatin-1 may also have a central or peripheral role in the initiation of pubertal process and may be related to PT pathogenesis.

Experimental validation of a novel compact focusing scheme for future energy-frontier linear lepton colliders.

A novel scheme for the focusing of high-energy leptons in future linear colliders was proposed in 2001 [P. Raimondi and A. Seryi, Phys. Rev. Lett. 86, 3779 (2001)]. This scheme has many advantageous properties over previously studied focusing schemes, including being significantly shorter for a given energy and having a significantly better energy bandwidth. Experimental results from the ATF2 accelerator at KEK are presented that validate the operating principle of such a scheme by demonstrating the demagnification of a 1.3 GeV electron beam down to below 65 nm in height using an energy-scaled version of the compact focusing optics designed for the ILC collider.

Lower cocaine- and amphetamine-regulated transcript levels pose a risk for developing PCOS.

UNLABELLED: Cocaine- and amphetamine-regulated transcript (CART) is an anorectic neuropeptide abundantly expressed in the central, peripheral, and enteric nervous systems, as well as in several different endocrine cell types. Besides regulating food intake and endocrine function, it is also proposed to modulate ovarian function during follicular waves in cattle and has potent inhibitory effects on follicular development. Polycystic ovary syndrome (PCOS), presenting itself with multiple follicular ovarian cysts, is the most common endocrinological disorder among women of reproductive age. Here we aimed to investigate the association of this peptide with PCOS. Our research was designed as a case-control study, in which a total of 148 subjects (73 with PCOS and 75 age- and BMI-matched CONTROLS) were consecutively recruited. Fasting blood glucose (FBG), insulin, high-sensitivity C-reactive protein, lipids, follicle stimulating hormone, luteinizing hormone, estradiol, CART, and free testosterone levels were measured in all participants. Homeostasis model assessment of insulin resistance (HOMA-IR) and body mass index (BMI) were calculated. CART levels were found to be significantly lower in patients with PCOS (PCOS: 90.77 ± 5.98 pg/ml, CONTROLS: 93.24 ± 8.17 pg/ml, p=0.038). Pearson's correlation analysis showed that CART was significantly and negatively correlated with BMI and waist circumference in both (PCOS and control) groups. In CONTROLS only, CART was positively correlated with insulin and HOMA-IR, and negatively correlated with FBG. Logistic regression analysis results are suggestive of a possible protective effect of CART against PCOS (OR: 0.94, 95% CI=0.888-0.997, p=0.038).

Altered activity of lysophospholipase D, which produces bioactive lysophosphatidic acid and choline, in serum from women with pathological pregnancy.

Altered lipid metabolism is associated with human abnormal pregnancy, such as pre-eclampsia and preterm labor, and potentially leads to fetus loss. A causative factor for the onset and progress of the systemic multifactorial syndromes associated with the pathological pregnancy is oxidized low-density lipoprotein, an active identity of which was postulated to be lysophosphatidic acid (LPA). We previously found that LPA is produced extracellularly by plasma lysophospholipase D (lysoPLD) activity of autotaxin, a tumor cell motility-stimulating protein. In this study, a convenient assay based on the choline released from endogenous substrate or exogenous lysophosphatidylcholine (LPC) was used for comparison of serum lysoPLD activity among patients with normal and abnormal pregnancy. The serum choline-producing activity was found to be mainly due to autotaxin, and dependent on its dilution rate. There was some association between low dilution dependency of serum lysoPLD activity toward an exogenous LPC and high lysoPLD activity toward endogenous substrates in cases of patients with preterm labor and pre-eclampsia. However, there was no difference in the serum level of LPC between women with normal pregnancy and those with pathological pregnancy. These results indicate that production of bioactive LPA by lysoPLD activity is elevated by an unknown mechanism that may be related to increased availability of endogenous substrates LPC, but not its concentration in human serum. If the level of LPA in blood circulation is elevated in the pathological pregnancies in vivo, it may play a role in induction and/or progression of systemic vascular dysfunction seen patients with preterm labor or pre-eclampsia.

Cocktail-substrate assay system for mechanism-based inhibition of CYP2C9, CYP2D6, and CYP3A using human liver microsomes at an early stage of drug development.

We established a mechanism-based inhibition cocktail-substrate assay system using human liver microsomes and drug-probe substrates that enabled simultaneous estimation of the inactivation of main cytochrome P450 (CYP) enzymes, CYP2C9, CYP2D6, and CYP3A, in drug metabolism. The inactivation kinetic parameters of typical mechanism-based inhibitors, tienilic acid, paroxetine, and erythromycin, for each enzyme in the cocktail-substrate assay were almost in agreement with the values obtained in the single-substrate assay. Using this system, we confirmed that multiple CYP inactivation caused by mechanism-based inhibitors such as isoniazid and amiodarone could be detected simultaneously. Mechanism-based inhibition potency can be estimated by the determination of the observed inactivation rate constants (k(obs)) at a single concentration of test compounds because the k(obs) of eleven CYP3A inactivators at 10 microM in the assay system nearly corresponded to k(inact)/K(I) values, an indicator of a compound's propensity to alter the activity of a CYP in vivo (R(2) = 0.97). Therefore, this cocktail-substrate assay is considered to be a powerful tool for evaluating mechanism-based inhibition at an early stage of drug development.

Thrombin induces striatal neurotoxicity depending on mitogen-activated protein kinase pathways in vivo.

Intracerebral hemorrhage represents stroke characterized by formation and expansion of hematoma within brain parenchyma. Blood-derived factors released from hematoma are considered to be involved in poor prognosis of this disorder. We previously reported that thrombin, a blood-derived serine protease, induced cytotoxicity in the cerebral cortex and the striatum in organotypic slice cultures, which depended on mitogen-activated protein kinase (MAPK) pathways. Here we investigated the mechanisms of thrombin cytotoxicity in the striatum in vivo. Thrombin microinjected into the striatum of adult rats induced neuronal death and microglial activation around the injection site. Neuronal loss without any sign of nuclear fragmentation was observed as early as 4 h after thrombin injection, which was followed by gradual neuronal death exhibiting nuclear fragmentation. Thrombin-induced damage assessed at 72 h after injection was partially but significantly reduced by concomitant administration of inhibitors of MAPK pathways. Activation of extracellular signal-regulated kinase (ERK) and p38 MAPK in response to thrombin was verified by Western blot analysis. Moreover, phosphorylated ERK and p38 MAPK were localized prominently in reactive microglia, and inhibition of microglial activation by minocycline attenuated thrombin-induced damage, suggesting that reactive microglia were responsible for thrombin-induced neuronal death. Thus, MAPK pathways and microglial activation may serve as therapeutic targets of pathogenic conditions associated with hemorrhagic stroke.

Minimal phenotype of mice homozygous for a null mutation in the forkhead/winged helix gene, Mf2.

Mf2 (mesoderm/mesenchyme forkhead 2) encodes a forkhead/winged helix transcription factor expressed in numerous tissues of the mouse embryo, including paraxial mesoderm, somites, branchial arches, vibrissae, developing central nervous system, and developing kidney. We have generated mice homozygous for a null mutation in the Mf2 gene (Mf2(lacZ)) to examine its role during embryonic development. The lacZ allele also allows monitoring of Mf2 gene expression. Homozygous null mutants are viable and fertile and have no major developmental defects. Some mutants show renal abnormalities, including kidney hypoplasia and hydroureter, but the penetrance of this phenotype is only 40% or lower, depending on the genetic background. These data suggest that Mf2 can play a unique role in kidney development, but there is functional redundancy in this organ and other tissues with other forkhead/winged helix genes.

Effect of c-myc gene expression on early inducible reactions required for erythroid differentiation in vitro.

By employing cell fusion between two genetically marked mouse erythroleukemia (MEL) cells in which an artificially introduced c-myc gene had been placed under the control of human metallothionein promoter, we investigated the mechanism of the suppressive action of c-myc gene expression in erythroid differentiation. The results indicated that the expression of the c-myc gene blocked the induction of dimethyl sulfoxide-inducible activity, one of the two early activities required for triggering the differentiation.

Development of high-order harmonic focusing system based on ellipsoidal mirror.

We have developed a focusing system for extreme ultraviolet light produced by high-order harmonic generation. An ellipsoidal mirror with a precise surface shape was fabricated and installed into the focusing system. A rigid mirror manipulator and a beam profiler were employed to perform precise and stable mirror alignment. As a demonstration of the focusing performance, high-order harmonics in the wavelength range of 13.5-19.5 nm were successfully focused into a 2.4 × 2.3 µm(2) spot.

Functional molecular size of trypsin inhibitors as determined by radiation inactivation analysis.

The molecular sizes of trypsin inhibitors obtained by radiation inactivation were investigated in relation to the functional domain size. The molecular weights obtained were 10,200 +/- 700 for ovomucoid, 17,800 +/- 400 for ovoinhibitor and 16,400 +/- 500 for soybean trypsin inhibitor. These values mostly agreed with the size of domain which has trypsin inhibitory activity, suggesting that the radiation inactivation analysis indicates the minimum functional domain size.

Processing and activation of recombinant mouse mastocytoma histidine decarboxylase in the particulate fraction of Sf9 cells by porcine pancreatic elastase.

Mature 53 kDa histidine decarboxylase (HDC) peptide is produced from a precursor 74 kDa peptide. The mechanism of specific cleavage by processing enzyme is unknown. Using the recombinant mouse 74 kDa HDC, we found that porcine pancreatic elastase specifically converted the inactive 74 kDa HDC to its active form of 53 kDa HDC.

Probability that the commitment of murine erythroleukemia cell differentiation is determined by the c-myc level.

During the commitment of mouse erythroleukemia cell differentiation, c-myc mRNA levels change dramatically. To examine the involvement of c-myc in the commitment of these cells, we have introduced the rat c-myc gene driven by inducible, heterologous (human metallothionein IIA) gene promoter into murine erythroleukemia cells and we have examined the ability of the transformed cells to undergo commitment to terminal differentiation. The induction of the exogenous c-myc gene expression inhibited the commitment of these cells. Time-dependent inhibition of the commitment was observed with the addition of zinc at an appropriate time after the induction with dimethyl sulfoxide. The result clearly indicated that late decline, not early decline, is required for the commitment. By examining the transformants expressing the exogenous c-myc mRNA at different levels, and the induction of the exogenous c-myc mRNA by varying the concentration of zinc, we demonstrated that the commitment may be determined by a stoichiometric amount of c-myc in the defined period. The data also suggest that the probability value for the commitment process occurring in a stochastic manner is well-correlated with the amount of c-myc mRNA.

CYP2D6 is the principal cytochrome P450 responsible for metabolism of the histamine H1 antagonist promethazine in human liver microsomes.

To determine which cytochrome P450 form is involved in the promethazine [10-(2-dimethylaminopropyl) phenothiazine] metabolism, in vitro analysis using human liver microsomes were performed. Promethazine was mainly biotransformed to ring-hydroxylated, S-oxidized and N-demethylated metabolites. The promethazine hydroxylase in human liver microsomes was inhibited by SKF-525A, propranolol, sparteine, quinidine and anti-CYP2D6 serum suggesting involvement of a P450 related to CYP2D6. Lineweaver-Burk plots for the hydroxylation, S-oxidation and N-demethylation indicated that the hydroxylation occurred with a low K(m) value in human liver microsomes. Microsomes from genetically-engineered human B-lymphoblastoid cells expressing CYP2D6 hydroxylated promethazine most efficiently as compared to other P450 forms, indicating that it was the principal P450 responsible for the metabolism of promethazine in human liver microsomes. The inhibition of CYP2D6-catalysed bufuralol 1'-hydroxylase by various histamine H3 antagonists including promethazine suggested that promethazine and some other histamine H1 antagonists could be inhibitors of this P450 in human liver microsomes.

Characterization of a novel variant (S145C/L311V) of 3alpha-hydroxysteroid/dihydrodiol dehydrogenase in human liver.

Human liver 3alpha-hydroxysteroid/dihydrodiol dehydrogenase (DD) is involved in the metabolism of steroid hormones and polycyclic aromatic hydrocarbons, and is also responsible for the reduction of ketone-containing drugs. To account for the interindividual difference in the activity, we isolated and characterized clones for the human liver enzymes. The sequence of the cDNA clone coding for the variant differed from that coding for the wild-type DD by two nucleotides (substitutions of C with G at positions 434 and 931) which caused two amino acid replacements, Ser145 to Cys (S145C) and Leu311 to Val (L311V). The heterologous expression of the variant mRNA was confirmed in four of 31 liver samples from Japanese by an allele-specific polymerase chain reaction. The effects of the mutations on the catalytic properties were examined with the recombinant enzymes expressed in Escherichia coli. The introduction of S145C/L311V double mutations resulted in three- to five-fold decreased activities for xenobiotic and steroidal substrates, whereas no significant change was observed by an introduction of the S145C mutation alone. The results substantiate the existence of polymorphic forms for human liver DD, and also suggest the importance of the residue at position 311 for substrate binding to the enzyme.

Minimal functional size of porcine lung and testicular angiotensin-converting enzymes deduced from radiation inactivation analysis. Interaction of two highly homologous domains in somatic isoenzyme.

Domain structures of porcine lung and testicular angiotensin-converting enzymes (ACE) were studied by radiation inactivation to test the hypothesis that lung ACE has two catalytic sites localized to discrete, structurally independent domains (the N- and C-domains) of approximately equal size. The minimum functional sizes of lung and testicular ACE, calculated from the inactivation curves obtained, were 140 and 74 kDa, respectively. Since testicular ACE has been demonstrated to contain only the C domain, this result indicates that the two domains in lung ACE are not independent but are, in fact, structurally tightly linked.

Induction of specific protein tyrosine phosphatase transcripts during differentiation of mouse embryonal carcinoma (F9) cells.

We have investigated the pattern of PTPase transcript expression during in vitro differentiation of mouse embryonal carcinoma (F9) cells. While the transcripts of most PTPases were unchanged or undetected during embryonal differentiation induced by retinoic acid, several PTPase transcripts exhibited distinct patterns of induction. Mutant cells defective in differentiation did not display the induction of some of these PTPase transcripts. Interestingly, three out of the four PTPase transcripts induced were the same PTPase transcripts induced during in vitro erythroid differentiation of mouse erythroleukemia (MEL) cells [(1994) J. Biol. Chem. 269, 4709-4712] [corrected]. The possible role played by specific PTPases in cell differentiation is discussed.

Characterization of a protein tyrosine phosphatase (RIP) expressed at a very early stage of differentiation in both mouse erythroleukemia and embryonal carcinoma cells.

From our previous studies, several protein tyrosine phosphatases (PTPase) are implicated in the early events leading to in vitro differentiation of both mouse erythroleukemia (MEL) and embryonal carcinoma (F9) cells. Among the PTPases, recent experiments suggest that a new PTPase (RIP) plays a critical role in differentiation processes, particularly at their early stages. We isolated cDNA clones for RIP from a RNA preparation isolated from differentiating MEL cells, and determined the total 7932 bp base sequence for RIP cDNA. The cDNA codes for a putative 269.8 kDa (2450 amino acids) protein with a PTPase catalytic domain. We have demonstrated that the transcripts exist in multiple forms, and among mouse tissues they were found predominantly in kidney and, to a lesser extent, in lung, heart, brain and testis. The RIP gene was mapped between D5Mit90 and D5Mit25 on mouse chromosome 5.

Localization of fractalkine and CX3CR1 mRNAs in rat brain: does fractalkine play a role in signaling from neuron to microglia?

Localization of the mRNAs for fractalkine, a CX3C chemokine, and for its receptor CX3CR1 was investigated in the rat brain. In situ hybridization study revealed that fractalkine mRNA was dominantly expressed in neuronal cells particularly in the olfactory bulb, cerebral cortex, hippocampus, caudate putamen and nucleus accumbens. In vitro study using enriched neuronal or glial culture supported the dominant expression of fractalkine mRNA in neurons. On the other hand, CX3CR1 mRNA was dominantly expressed in glial cells throughout the whole brain. The in vitro study suggested the cells expressing CX3CR1 mRNA are microglia, not astrocytes or neurons. Fractalkine appears to function as a signal molecule from neuron to microglia.

Protective effects of 1 alpha,25-(OH)(2)D(3) against the neurotoxicity of glutamate and reactive oxygen species in mesencephalic culture.

This study was undertaken to determine whether 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)(2)D(3)], an active metabolite of vitamin D, protects dopaminergic neurons against the neurotoxic effects of glutamate and dopaminergic toxins using rat mesecephalic culture. Brief glutamate exposure elicited cytotoxicity in both dopaminergic and non-dopaminergic neurons. Pretreatment, but not co-administration, of 1 alpha,25-(OH)(2)D(3) protected both types of neurons against the cytotoxicity of glutamate in a concentration- and time-dependent manner. The neuroprotective effect of 1 alpha,25-(OH)(2)D(3) was inhibited by the protein synthesis inhibitor, cycloheximide. To investigate the mechanisms of these neuroprotective effects, we examined the effects of 1 alpha,25-(OH)(2)D(3) on neurotoxicity induced by calcium ionophore and reactive oxygen species (ROS). Pretreatment with 1 alpha,25-(OH)(2)D(3) protected both types of neurons against the cytotoxicity induced by A23187 in a concentration-dependent manner. Furthermore, 24-h pretreatment with 1 alpha,25-(OH)(2)D(3) concentration-dependently protected both types of neurons from ROS-induced cytotoxicity. A 24-h incubation with 1 alpha,25-(OH)(2)D(3) inhibited the increase in intracellular ROS level following H(2)O(2) exposure. A 24-h exposure to 1-methyl-4-phenylpyridium ion (MPP(+)) or 6-hydroxydopamine (6-OHDA) exerted selective neurotoxicity on dopaminergic neurons, and these neurotoxic effects were ameliorated by 1 alpha,25-(OH)(2)D(3). These results suggest that 1 alpha,25-(OH)(2)D(3) provides protection of dopaminergic neurons against cytotoxicity induced by glutamate and dopaminergic toxins by facilitating cellular functions that reduce oxidative stress.