Arglabin [1(R),10(S)-epoxy-5(S),5(S),7(S)-guaia-3(4),11(13)-dien-6, 12-olide], a sesquiterpene gamma-lactone is isolated from Artemisia glabella, a species of wormwood endemic to the Karaganda region of Kazakstan. The compound has been modified to render it water-soluble through addition of a dimethylaminohydrochloride group to the C(13) carbohydride moiety to yield Arglabin-DMA. Arglabin-DMA is a registered antitumor substance in the Republic of Kazakstan. Previously, we have shown that this compound prevents protein farnesylation without altering geranylgeranylation. We now report that Arglabin-DMA inhibits the incorporation of [(3)H]farnesylpyrophosphate into human H-ras protein by FTase with an IC(50) of no greater than 25 microM. Kinetic studies show that the phosphorylated form of this compound competitively inhibits the binding of farnesyl diphosphate to FTase. This mechanism of action is different from other reported peptidomimetic FTIs which lower the affinity of ras protein to FTase. Our in vitro studies confirm that Arglabin-DMA inhibits post-translational modification of ras protein in cells. Arglabin-DMA inhibits anchorage-dependent proliferation of NB cells (IC50=10 microg/ml) and inhibits anchorage-independent growth of NB and KNRK cells with about the same IC(50). Soft-agar colony formation assay of H-ras and K-ras transformed cells show IC(50)s to be 2 and 5 microg/ml, respectively. In primary cultures of human tumor cells, Arglabin-DMA inhibits cell proliferation of a variety of tumor types with IC(90)s in the range of 0.85 to 5.0 microg/ml. Because of these pharmacologic properties, we propose that Arglabin-DMA is suitable for the treatment of ras related malignancies.

UNASSIGNED

Pulmonary vein isolation (PVI) using contact force (CF) sensing ablation catheters currently relies on CF and force-time integral (FTI) guidelines. Such measurement of lesion effectiveness still lacks information on current delivery to the tissue, influenced by system impedance and power. Lesion Index (LSI) is a multi-parametric index incorporating CF and radiofrequency current data across time. We aimed to prospectively assess the efficacy of an LSI-guided approach to PVI in patients with paroxysmal atrial fibrillation (PAF).

UNASSIGNED

The study prospectively enrolled 28 consecutive patients with PAF undergoing PVI with a CF sensing catheter (TactiCathTM, Abbott). LSI-guided ablation target was adapted according to the mean regional thickness of pulmonary vein antra (PVA): LSI range 5.5-6 was pursued in the anterior and septal portions of PVA, 5-5.5 elsewhere. Data from 32 consecutive PAF patients who underwent PVI ablation with a non-CF guided approach (NCF-group) were retrospectively collected for comparison of procedural and clinical outcome.AF-free survival rate at follow-up (17±6 months) was higher for LSI-guided group than NCF-group (89.3% vs 65.6%, p=0.037), with no increase in periprocedural complication rate (no tamponades or other major adverse events reported). Among 1126 lesions with LSI within target range (5-6), average CF was >10g and <30g for 976 lesions (86.7%). Moreover, 1015 lesions (90.1%) had FTI)400gs, but with wide distribution: 30.2% within 400-500gs, 30.0% within 501-600gs, 29.9% over 600gs.

UNASSIGNED

In this first prospective study, LSI-guided PVI improved clinical outcome without any increase in complication rate when compared with standard, non-LSI-guided approach.

OBJECTIVE

Late preterm infants (LPIs) (gestation 34 weeks and 0 days to 36 weeks and 6 days) compared with full-term infants (FTIs) are at increased risk for mortality and short- and long-term morbidity. The objective of this study was to assess the neurodevelopmental outcomes in a longitudinal cohort study of LPIs from infancy to school age and determine predictive values of earlier developmental testing compared with school-age testing.

METHODS

We used general estimating equations to calculate the odds of school readiness in a nationally representative cohort of 4900 full-term and 950 late preterm infants. We generated positive and negative predictive values of the ability of the 24-month Mental Developmental Index (MDI) scores of the Bayley Short Form, Research Edition, to predict Total School Readiness Score (TSRS) at kindergarten age.

RESULTS

In multivariable analysis, late preterm infants had higher odds of worse TSRSs (adjusted odds ratio 1.52 [95% confidence interval 1.06-2.18], P = .0215). The positive predictive value of a child having an MDI of <70 at 24 months and a TSRS <5% at kindergarten was 10.4%. The negative predictive value of having an MDI of >70 at 24 months and a TSRS >5% was 96.8%. Most infants improved score ranking over the study interval.

CONCLUSIONS

LPIs continue to be delayed at kindergarten compared with FTIs. The predictive validity of having a TSRS in the bottom 5% given a MDI <70 at 24 months was poor. A child who tested within the normal range (>85) at 24 months had an excellent chance of testing in the normal range at kindergarten.

OBJECTIVE

Catheter-tissue contact is critical for effective lesion creation in radiofrequency catheter ablation (RFCA). In a multicenter prospective study, we assessed the relationship between catheter contact force (CF) during RFCA for paroxysmal atrial fibrillation (AF) and clinical recurrences over a mid-term follow-up.

METHODS

All patients underwent RFCA for paroxysmal AF by antral pulmonary vein (PV) isolation, aiming at entry and exit conduction block in all PVs. A new open-irrigated tip catheter with CF sensing (SmartTouch(TM), Biosense Webster Inc. CA) was used. All patients were followed for at least 12 months and the relationship between CF and clinical outcomes assessed.

RESULTS

One year follow-up was available in 92/95 of the patients enrolled. Acute PV isolation was achieved in 100 % of the veins. Mean CF during RFCA was 12.2 ± 3.9 g. Mean force-time integral (FTI) was 733 ± 505 gs. Following the 3-month blanking period, 17 (18 %) patients experienced at least 1 atrial tachyarrhythmia relapse. There was no statistical difference in mean CF (13 ± 3.4 g vs 12 ± 4 g, p = 0.32) and mean FTI (713 ± 487 gs vs 822 ± 590 gs, p = 0.42) between patients with and without arrhythmia recurrences. Recurrences were recorded in 22 % of patients achieving a mean FTI value below the median of 544 gs and in 15 % of patients with a mean FTI value above the median (p = 0.64).

CONCLUSIONS

RFCA with CF data during PV isolation for paroxysmal AF improves physician's knowledge on catheter-tissue contact. In the present dataset, however, higher CF values did not impact mid-term clinical RFCA outcome.

Purpose: Mutant KRAS is a major driver of pancreatic oncogenesis and therapy resistance, yet KRAS inhibitors are lacking in the clinic. KRAS requires farnesylation for membrane localization and cancer-causing activity prompting the development of farnesyltransferase inhibitors (FTIs) as anticancer agents. However, KRAS becomes geranylgeranylated and active when cancer cells are treated with FTIs. To overcome this geranylgeranylation-dependent resistance to FTIs, we designed FGTI-2734, a RAS C-terminal mimetic dual FT and geranylgeranyltransferase-1 inhibitor (GGTI).Experimental Design: Immunofluorescence, cellular fractionation, and gel shift assays were used to assess RAS membrane association, Western blotting to evaluate FGTI-2734 effects on signaling, and mouse models to demonstrate its antitumor activity.Results: FGTI-2734, but not the selective FTI-2148 and GGTI-2418, inhibited membrane localization of KRAS in pancreatic, lung, and colon human cancer cells. FGTI-2734 induced apoptosis and inhibited the growth in mice of mutant KRAS-dependent but not mutant KRAS-independent human tumors. Importantly, FGTI-2734 inhibited the growth of xenografts derived from four patients with pancreatic cancer with mutant KRAS (2 G12D and 2 G12V) tumors. FGTI-2734 was also highly effective at inhibiting, in three-dimensional cocultures with resistance promoting pancreatic stellate cells, the viability of primary and metastatic mutant KRAS tumor cells derived from eight patients with pancreatic cancer. Finally, FGTI-2734 suppressed oncogenic pathways mediated by AKT, mTOR, and cMYC while upregulating p53 and inducing apoptosis in patient-derived xenografts in vivoConclusions: The development of this novel dual FGTI overcomes a major hurdle in KRAS resistance, thwarting growth of patient-derived mutant KRAS-driven xenografts from patients with pancreatic cancer, and as such it warrants further preclinical and clinical studies.

Of the three RAS oncoproteins, only HRAS is delocalized and inactivated by farnesyltransferase inhibitors (FTI), an approach yet to be exploited clinically. In this study, we treat mice bearing Hras-driven poorly differentiated and anaplastic thyroid cancers (Tpo-Cre/HrasG12V/p53flox/flox ) with the FTI tipifarnib. Treatment caused sustained tumor regression and increased survival; however, early and late resistance was observed. Adaptive reactivation of RAS-MAPK signaling was abrogated in vitro by selective RTK (i.e., EGFR, FGFR) inhibitors, but responses were ineffective in vivo, whereas combination of tipifarnib with the MEK inhibitor AZD6244 improved outcomes. A subset of tumor-bearing mice treated with tipifarnib developed acquired resistance. Whole-exome sequencing of resistant tumors identified a Nf1 nonsense mutation and an activating mutation in Gnas at high allelic frequency, supporting the on-target effects of the drug. Cell lines modified with these genetic lesions recapitulated tipifarnib resistance in vivo This study demonstrates the feasibility of targeting Ras membrane association in cancers in vivo and predicts combination therapies that confer additional benefit.Significance: Tipifarnib effectively inhibits oncogenic HRAS-driven tumorigenesis and abrogating adaptive signaling improves responses. NF1 and GNAS mutations drive acquired resistance to Hras inhibition, supporting the on-target effects of the drug. Cancer Res; 78(16); 4642-57. ©2018 AACR.

Thyroid function was evaluated in 20 patients aged 8-30 years who were suffering from homozygous beta-thalassaemia. All patients have been receiving frequent blood transfusions and treated for the resulting transfusional iron overload with intramuscular injections of Desferrioxamine. Total thyroxine (T4), T3-uptake, total triiodothyronine (T3), and reverse triiodothyronine (rT3) were measured. In addition a standard TRH stimulation test was performed and blood samples were checked for the presence of thyroid antibodies. It was found that total T4 was significantly lower in the patients than in the controls. Total T3 and rT3 levels were similar in both patients and controls and all patients were negative for thyroid antibodies. T3 uptake in the patients was also statistically different from the controls resulting in significantly lower free thyroxine index (FT1). Basal TSH values were not different from the controls but the TSH increase following TRH stimulation was significantly higher in the patients suggesting, together with the low total T4 and FTI, a state of compensated hypothyroidism.

Postoperative thyroid status was classified into 6 categories on the basis of serum free thyroxine index (FTI), serum triiodothyronine (T-3) and serum TSH concentration. Review of 325 patients who underwent thyroidectomy for Graves' disease more than 4 years previously showed that 25 patients (7.7 per cent) had recurrent hyperthyroidism. Six patients (1.8 per cent) were classified as equivocal hyperthyroid since either FTI or T-3 was above the normal range. Two hundred and twenty-two patients (68.3 per cent) were unequivocally euthyroid and 33 patients (10.3 per cent) were euthyroid with elevated TSH levels. Twenty patients (6.2 per cent) were equivocal hypothyroid since either their FTI or T-3 values were below the normal range and TSH were increased. Hypothyroidism was present in 19 patients (5.8 per cent), of whom 11 had no clinical manifestation of thyroid dysfunction. The incidence of hypothyroidism did not correlate with the intervals between operation and review. The second review, performed in 189 patients 18 months after the first, showed that there was a change in thyroid functional status in 46 patients, of whom 32 had an increased level of function, including one hypothyroid and 7 equivocal hypothyroid patients who became euthyroid spontaneously. Thus postoperative hypothyroidism in some patients can recover without medication. Also thyroid function in some postoperative patients is not maintained at a fixed level but may fluctuate.

This review analyses what is known concerning the immune response of preterm (PTIs) and low birth weight infants (LBWIs) to widely used vaccines, the protection they receive from routine immunisation, and the safety and tolerability of the vaccines themselves. It shows why PTIs and LBWIs should be vaccinated using the same schedules as those usually recommended for full-term infants (FTIs), except in the case of hepatitis B vaccine, whose schedule should be repeated in infants who received the first dose during the first days of life when they weighed less than 2000 g because of their reduced immune response. Vaccines are immunogenic, safe and well tolerated in PTIs and LBWIs, in whom early active immunisation is particularly important because they are among the most vulnerable subjects for pediatric infectious diseases. It is therefore essential to make every effort to convince pediatricians and parents that compliance with these recommendations will not cause any clinical problems.

Mutants of lamin A cause diseases including the Hutchinson-Gilford progeria syndrome (HGPS) characterized by premature aging. Lamin A undergoes a series of processing reactions, including farnesylation and proteolytic cleavage of the farnesylated C-terminal domain. The role of cleavage is unknown but mutations that affect this reaction lead to progeria. Here we show that interphase serine 22 phosphorylation of endogenous mutant lamin A (progerin) is defective in cells from HGPS patients. This defect can be mimicked by expressing progerin in human cells and prevented by inhibition of farnesylation. Furthermore, serine 22 phosphorylation of non-farnesylated progerin was enhanced by a mutation that disrupts lamin A head to tail interactions. The phosphorylation of lamin A or non-farnesylated progerin was associated to the formation of spherical intranuclear lamin A droplets that accumulate protein kinases of the CDK family capable of phosphorylating lamin A at serine 22. CDK inhibitors compromised the turnover of progerin, accelerated senescence of HGPS cells and reversed the effects of FTI on progerin levels. We discuss a model of progeria where faulty serine 22 phosphorylation compromises phase separation of lamin A polymers, leading to accumulation of functionally impaired lamin A structures.

Atherosclerotic cardiovascular disease is the number one cause of death for adults in Western society. Plasminogen activator inhibitor-1 (PAI-1), the major physiological inhibitor of plasminogen activators, has been implicated in both thrombogenesis and atherogenesis. Previous studies demonstrated that copper-oxidized low-density lipoprotein (C-oLDL) stimulated production of PAI-1 in vascular endothelial cells (EC). The present study examined the involvement of lectin-like oxidized LDL receptor-1 (LOX-1) and Ras/Raf-1/ERK1/2 pathway in the upregulation of PAI-1 in cultured EC induced by oxidized LDLs. The results demonstrated that C-oLDL or FeSO(4)-oxidized LDL (F-oLDL) increased the expression of PAI-1 or LOX-1 in human umbilical vein EC (HUVEC) or coronary artery EC (HCAEC). Treatment with C-oLDL significantly increased the levels of H-Ras mRNA, protein, and the translocation of H-Ras to membrane fraction in EC. LOX-1 blocking antibody, Ras farnesylation inhibitor (FTI-277), or small interference RNA against H-Ras significantly reduced C-oLDL or LDL-induced expression of H-Ras and PAI-1 in EC. Incubation with C-oLDL or F-oLDL increased the phosphorylation of Raf-1 and ERK1/2 in EC compared with LDL or vehicle. Treatment with Raf-1 inhibitor blocked Raf-1 phosphorylation and the elevation of PAI-1 mRNA level in EC induced by C-oLDL or LDL. Treatment with PD-98059, an ERK1/2 inhibitor, blocked C-oLDL or LDL-induced ERK1/2 phosphorylation or PAI-1 expression in EC. The results suggest that LOX-1, H-Ras, and Raf-1/ERK1/2 are implicated in PAI-1 expression induced by oxidized LDLs or LDL in cultured EC.

In this study we contrasted the Category fluency and Letter fluency performance of 198 normal subjects, 57 Alzheimer's patients and 57 patients affected by traumatic brain injury (TBI). The aim was to check whether, besides the prevalence of Category fluency deficit often reported among Alzheimer's patients, the TBI group presented the opposite dissociation. According to some recent claims, in fact, the deficit of TBI would be equally severe for both fluency types. The inquiry followed different approaches for data analysis, including the evaluation of a unique index (Fluency Type Index or FTI), independent of the overall fluency and aimed at expressing at individual subject level the relationship between Category and Letter fluency. The results confirmed that Alzheimer's patients are more defective on Category than Letter fluency, and also clearly indicated that an opposite pattern applies to TBI patients. TBI seems to cause a relatively more severe impairment of Letter than Category fluency, probably due to its impact on the frontal lobe structures. We discuss whether, on the basis of the statistical distribution of our data, it is worth considering as homogeneous populations broadly defined groups as Alzheimer's or TBI patients.

BACKGROUND

Overweight and obesity are increasing health problems that are not restricted to adults only. Childhood obesity is associated with metabolic, psychological and musculoskeletal comorbidities. However, knowledge about the effect of obesity on the foot function across maturation is lacking. Decreased foot function with disproportional loading characteristics is expected for obese children. The aim of this study was to examine foot loading characteristics during gait of normal-weight, overweight and obese children aged 1-12 years.

METHODS

A total of 10382 children aged one to twelve years were enrolled in the study. Finally, 7575 children (m/f: n = 3630/3945; 7.0±2.9 yr; 1.23±0.19 m; 26.6±10.6 kg; BMI: 17.1±2.4 kg/m2) were included for (complete case) data analysis. Children were categorized to normal-weight (≥3rd and <90th percentile; n = 6458), overweight (≥90rd and <97th percentile; n = 746) or obese (>97th percentile; n = 371) according to the German reference system that is based on age and gender-specific body mass indices (BMI). Plantar pressure measurements were assessed during gait on an instrumented walkway. Contact area, arch index (AI), peak pressure (PP) and force time integral (FTI) were calculated for the total, fore-, mid- and hindfoot. Data was analyzed descriptively (mean ± SD) followed by ANOVA/Welch-test (according to homogeneity of variances: yes/no) for group differences according to BMI categorization (normal-weight, overweight, obesity) and for each age group 1 to 12 yrs (post-hoc Tukey Kramer/Dunnett's C; α = 0.05).

RESULTS

Mean walking velocity was 0.95 ± 0.25 m/s with no differences between normal-weight, overweight or obese children (p = 0.0841). Results show higher foot contact area, arch index, peak pressure and force time integral in overweight and obese children (p<0.001). Obese children showed the 1.48-fold (1 year-old) to 3.49-fold (10 year-old) midfoot loading (FTI) compared to normal-weight.

CONCLUSIONS

Additional body mass leads to higher overall load, with disproportional impact on the midfoot area and longitudinal foot arch showing characteristic foot loading patterns. Already the feet of one and two year old children are significantly affected. Childhood overweight and obesity is not compensated by the musculoskeletal system. To avoid excessive foot loading with potential risk of discomfort or pain in childhood, prevention strategies should be developed and validated for children with a high body mass index and functional changes in the midfoot area. The presented plantar pressure values could additionally serve as reference data to identify suspicious foot loading patterns in children.

OBJECTIVE

Corneal fibroblast cell (CFC) reticulation is involved in the structural development of corneal stroma and in wound healing. In an earlier paper, it was reported that the expression of VEGFR-1 by CFCs is related to their reticulogenic properties in vitro and decreases with the age of the donors. The present study was focused on the nonreticulogenic corneal fibroblast population and explored whether these cells can be induced to form cell networks in vitro.

METHODS

The network formation was analyzed using an array of signaling pathway inhibitors: wortmannin for PI3 kinase, U0126 for MEK-1/2 kinase, Rottlerin for PKC, farnesyl transferase inhibitor (FTI-277) for Ras, and picropodophyllin (PPP) for IGFR-1. Among the several growth factors studied, IGF seemed to be crucial to cell network formation. The presence of IGF signaling was demonstrated using gene array analysis and was confirmed by RT-PCR and immunocytochemistry and by cell network formation on reduced synthetic basement membrane arrays. The pleiotropic effect of IGF-1 on the cells was analyzed by protein cytokine array.

RESULTS

The genesis of reticulation was found to occur via MEK-1/2 and IGFR pathways, since inhibitors of these signaling pathways reduced or prevented cell network formation. The addition of exogenous IGF-1 generated a cell network structure in corneal fibroblasts obtained from healthy donors, indicating the involvement of IGF-1.

CONCLUSIONS

IGF signaling and the MEK-1/2 pathway are involved in the cell network formation of corneal fibroblast cells from aged donors.

OBJECTIVE

Early exposure of preterm infants to visual stimulation could affect the process of visual maturation. The aim of this study was to assess the impact of prematurity on visual function at 15 months post-term.

METHODS

Visual function was assessed in 102 preterm (PTI) and 50 full-term infants (FTI) without major cerebral pathology or retinopathy of prematurity (ROP) of grade 2 to 5, at 15 months' corrected age. The visual acuity, refractive status, contrast sensitivity, strabismus, fundus, and neurodevelopment were examined.

RESULTS

Impairments of individual visual functions were 2 to 10 times more common in PTI than FTI. However, the difference was significant only for refractive errors (p = 0.007, odds ratio [OR] = 10.5). The incidence of visual deficits was higher in PTI with gestational age less than 32 weeks compared with PTI with higher gestational age (OR = 1.3 to 2.0), but not significantly. Of the PTI, 4.9%, 2.9%, and 10.8% had mild abnormalities on ultrasound scans, neuromotor, and developmental examination, respectively, which were not associated with increased incidence of visual deficits.

CONCLUSIONS

Premature exposure to visual stimulation does not induce visual maturation but it is associated with impairment of certain aspects of visual function even in the absence of major ROP or neurodevelopmental deficits.

Antidepressant drugs are among the most common drugs involved in fatal poisoning and large variations between antidepressant drugs have been noted. Despite the fact that a large number of studies have calculated a fatal toxicity index (FTI) for antidepressants, no serious attempts have been made to compare the differences in fatal toxicity against known pharmacological and toxicological differences in receptor affinity. It is potentially from such data that screening of drugs during their pre-clinical development can be facilitated. We examined correlations between the FTI and noradrenaline (norepinephrine)/serotonin (5-hydroxytryptamine; 5-HT) reuptake inhibition selectivity, the dose that is lethal to 50% of animals (LD50), lipid solubility, and antagonist activity at cholinergic, histaminergic, alpha-adrenergic and gamma-aminobutyric acid (GABA)A receptors or sodium and potassium channel blocking effects. We obtained data on the number of fatal poisonings between 1983 and 1992 in England and Wales caused by a single antidepressant drug from the Department of Health in the UK. This number was divided by the number of prescriptions in England for these drugs over this time to derive a FTI of deaths per million prescriptions. The highest FTIs were for amoxapine, viloxazine, desipramine and dothiepin. Lofepramine, paroxetine and fluoxetine had very low FTIs. Using Poisson regression, there was a significant positive relationship between the FTI of antidepressant drugs and their lethal toxicity in animals, and measures of their cardiac effects. The relative noradrenaline/serotonin reuptake inhibition, lipid solubility and their potency at histamine H1, muscarinic and alpha 1-adrenergic receptors had no substantial association with the FTI. Limited data suggest that some cardiac effects and potency as a GABAA antagonist may be important predictors of significant toxicity. Further data using standardised bio-assays are needed to compare the direct cardiac effects of antidepressants. Thus, the best current pre-clinical indicator of fatal toxicity in humans is the LD50 in animal studies. Clearly, there are humane and practical reasons for developing a better pre-clinical indicator of toxicity in overdose for this rapidly expanding group of drugs.

To delineate the incidence, clinical features, diagnosis, and treatment options for amiodarone induced hyperthyroidism (AIH), we reviewed the medical records of ten patients with AIH. Eight of these 10 patients were detected on initial review of the records of 200 patients in treatment with amiodarone, and the other 2 following notification by their endocrinologists. AIH occurred in 4.2% of patients being treated with amiodarone. At the time of diagnosis of AIH, the mean (SD) values for age, duration of treatment with amiodarone, and dose of amiodarone were 62.9 (8.96) years, 38.3 (20) months, and 366.7 (122) mg/day, respectively. The most common clinical features were weight loss and goiter (each seen in 90% of patients). Serum thyroxine (T4), triiodothyronine, and free thyroxine index (FTI) showed an increase of 84%, 47%, and 110%, while thyroid stimulating hormone (TSH) and resin T4 uptake decreased 96% and 14%, respectively, from previous values. The most consistent laboratory findings, seen in all patients, were subnormal TSH and abnormally high FTI. One patient required no treatment; another underwent prompt total thyroidectomy. The other eight were treated medically; two of them underwent total thyroidectomy later, for medical failure or adverse effects. Amiodarone was continued in four patients. The most commonly used antithyroid medication was propylthiouracil. AIH presents in the early or late phases of treatment with amiodarone with typical clinical features of a hyperthyroid state, fall in TSH, and increase in FTI. Antithyroid medications are reasonably effective in the management of AIH.

Methyl substitution at the 2-position of the imidazole ring greatly improved drug metabolism profiles, in human liver microsomes, of ras farnesyl-protein transferase inhibitor (FTI) candidates for drug development. Methyl substitution markedly reduced the P450 inhibitory potency of non-substituted FTIs for CYP3A4 (by a factor of 12-403) and 2C9 (by a factor of 4.2-28), while it had little effect on the CYP2D6 enzyme. An immunochemical inhibition study demonstrated that CYP3A4 plays a predominant role in the metabolism of both non-substituted and 2-methyl-substituted imidazole-containing FTI candidates. Very strong type II binding spectra with human liver microsomes were observed for all non-substituted FTIs, while methyl substitution markedly weakened type II spectra or shifted the type of spectra from II to I. This indicated that methyl substitution on the imidazole moiety interfered with the substrate-P450 heme interaction, likely due to a steric effect caused by the methyl group. A kinetics study revealed that the methyl substitution increased V(max) and K(m) values to the same extent. These studies suggested that the 2-methyl substitution on the imidazole ring improved its drug metabolism profile by reducing the potential to inhibit CYP3A4-mediated metabolism without affecting intrinsic metabolic clearance (V(max)/K(m)).

The TLR agonists, flagellin (FLG) and lipopolysaccharide (LPS) stimulate functional activation and cytokine gene expression via the extracellular signal regulated kinase 1/2 (ERK1/2) MAP kinase cascade. However, the upstream mechanisms of these signaling events remain unknown. In mammals, the small GTP-binding protein Ras mediates ERK1/2 activation through activation of downstream effectors Raf-1-MEK1/2-ERK1/2 in response to a variety of stimuli. It is not clear whether this classic Ras cascade plays a role in TLR signaling in avian cells. In the present study, we investigated the role of Ras in FLG- and LPS-mediated signaling in ERK activation in chicken heterophils. Treatment of heterophils with LPS caused a rapid (within 5min) activation of Ras-GTP. The role of Ras activation in LPS-induced stimulation of ERK1/2 was corroborated when the specific Ras inhibitor, FTI-277, inhibited ERK1/2 activation. The classic Ras-mediated pathway of ERK1/2 activation by LPS was confirmed when the specific Raf-1 inhibitor, GW 5074, and the MEK1/2 inhibitor, U0126, both reduced ERK activation by 51-60%. Of more interest was that treatment of the heterophils with FLG did not activate Ras-GTP. Likewise, neither FTI-277 nor GW 5074 had any effect on FLG-mediated activation of ERK1/2. Another small GTPase, Rap1, has been shown to play a role in mammalian neutrophil function. Using a Rap1-GTP pull-down assay, we found that FLG stimulation, but not LPS, of avian heterophils induced a rapid and transient Rap1 activation. Rap1 has been shown to activate the ERK1/2 via a different Raf family member B-Raf whose downstream effector is MEK1/2. We show here that FLG stimulation of heterophils induces the phosphorylation of Rap1. The FLG induction of the Rap1->>B-Raf->>MEK1/2->>ERK1/2 cascade was confirmed by the reduction of ERK1/2 activation by the specific Rap1 inhibitor (GGTI-298) and U0126. The results demonstrate that for the first time that the small GTPase Ras family is involved in TLR signaling of avian heterophils with the TLR agonists LPS (Ras) and FLG (Rap1) inducing differential signaling cascades to activate the downstream ERK MAP kinase.

BACKGROUND

Inhibitors of HMG-CoA reductase are widely used to prevent atherosclerosis progression. The expression of adhesion molecules on activated endothelial cells (EC) is an important step in the initiation and progression of atherosclerosis.

OBJECTIVE

We investigated whether adhesion molecule expression on activated EC is influenced by simvastatin, fluvastatin and pravastatin and, if so, by which mechanisms.

METHODS

Human EC from umbilical veins or saphenous veins were pretreated overnight with statins with or without mevalonate, and also for simvastatin or fluvastatin with the isoprenoid intermediates, farnesyl pyrophosphate (FPP), or geranylgeranyl pyrophosphate (GGPP). After 4-6 h activation with tumor necrosis factor (TNF)-alpha or lipopolysaccharide (LPS), surface adhesion molecule expression was evaluated by ELISA and by flow cytometry. The same experiments were performed with selective inhibitors of geranylgeranyltransferase (GGTI-286) and farnesyltransferase (FTI-277).

RESULTS

Pretreatment with simvastatin, fluvastatin or pravastatin potentiated the TNF-alpha and LPS-induced expression of E-selectin and VCAM-1, and mevalonate reversed the potentiating effect of these statins. GGPP also reversed the potentiating effect of simvastatin or fluvastatin on adhesion molecule expression, while FPP only partially reversed this effect. Furthermore, GGTI-286, but not FTI-277, mimicked the effect of simvastatin by increasing the TNF-alpha-mediated overexpression of E-selectin.

CONCLUSIONS

Statins increase E-selectin- and VCAM-1-induced expression on vascular endothelial cells stimulated with TNF-alpha or LPS. The inhibition of geranylgeranylated proteins could contribute to this effect.

The purpose of the present study was to evaluate the feasibility of partitioning myocardial O2 consumption (VO2) into mechanical and nonmechanical components in the whole heart preparation using a negative inotrope, 2,3-butanedione monoxime (BDM), which has been reported to have a selective effect on the contractile proteins in a low concentration range (< 6 mM). In six isolated bovine red blood cell-perfused rabbit hearts, VO2 and force-time integral (FTI) were measured during infusion of varying concentrations of BDM at a constant left ventricular volume chosen such that control left ventricular peak isovolumic pressure was approximately 100 mmHg. The VO2-FTI relation with BDM concentrations < or = 5 mM was highly linear (median r = 0.98). Its VO2-axis intercept at zero FTI had a positive value (mean 23% of control, 0.014 ml O2.beat-1 x 100 g-1). To confirm the selective effect of BDM on the contractile proteins, the intracellular free Ca2+ transient was measured with the fluorescent indicator indo 1 in three isolated buffer-perfused rabbit hearts. The amplitude of the Ca2+ transient was not altered by BDM at concentrations < or = 10 mM, although left ventricular developed pressure was markedly depressed. This finding indicates that BDM < or = 10 mM does not affect excitation-contraction coupling. We conclude that the VO2-axis intercept value of the VO2-FTI relation during BDM infusion in a low concentration range represents VO2 for nonmechanical energy utilization. The BDM method to partition VO2 into mechanical and nonmechanical components is thus feasible in the whole rabbit heart.

Osteoclast differentiation is a complex process involving cytoskeleton and nuclear reorganization. Osteoclasts regulate bone homeostasis and have a key role in bone degenerative processes. Osteolysis and osteoporosis characterize a subset of laminopathies, inherited disorders due to defects in lamin A/C. Laminopathies featuring bone resorption are characterized, at the molecular level, by anomalous accumulation of the unprocessed lamin A precursor, called prelamin A. To obtain a suitable cell model to study prelamin A effects on osteoclasts, prelamin A processing inhibitors FTI-277 or AFCMe were applied to peripheral blood monocytes induced to differentiate towards the osteoclastic lineage. Previous studies have shown that treatment with FTI-277 causes accumulation of non-farnesylated prelamin A, while AFCMe inhibition of prelamin A maturation causes accumulation of a farnesylated form. We demonstrate that monocytes subjected to FTI-277 treatment and mostly those subjected to AFCMe administration, differentiate towards the osteoclastic lineage more efficiently than untreated monocytes, in terms of number of multinucleated giant cells, mRNA expression of osteoclast-related genes and TRACP 5b activity. On the other hand, the bone resorption activity of osteoclasts obtained in the presence of high prelamin A levels is lower with respect to control osteoclasts. This finding may help the understanding of the osteolytic and osteoporotic processes that characterize progeroid laminopathies.

OBJECTIVE

To explore the possible effect of metformin on maternal and fetal androgens and antimüllerian hormone (AMH) levels at birth and to study the predictors of maternal and fetal AMH levels.

METHODS

Substudy of a randomized controlled trial (the PregMet study).

METHODS

University hospital.

METHODS

Women with polycystic ovary syndrome (PCOS) and their newborns (n = 132).

METHODS

Metformin, 2,000 mg/daily, or placebo from the first trimester until delivery.

METHODS

Androgens and AMH levels in maternal venous serum and in umbilical vein and artery serum.

RESULTS

Except for the increased free testosterone index (FTI) in the umbilical artery in boys, metformin did not influence maternal or fetal androgens, or AMH levels. The maternal body mass index (BMI) was a negative and FTI a strong positive predictor of maternal AMH levels. Maternal androgens and AMH levels did not correlate to fetal gender. In girls, gestational age, birth weight, or maternal androgens did not correlate to the AMH levels. In boys, birth weight was negatively correlated to the AMH levels.

CONCLUSIONS

Except for FTI, which was higher in boys, metformin had no impact on maternal or fetal androgen levels or the level of AMH. Fetal AMH, as a surrogate marker for ovarian development, was unaffected by maternal androgens. Birth weight and gestational age had no impact on AMH levels in girls; in boys, AMH probably reflects the physiologic variations due to birth weight.

BACKGROUND

NCT00159536 (www.clinicaltrials.gov).