The Comparative Bioavailability of Fluticasone and Azelastine Delivered as a Single Fixed Dose Combination (MP-AzeFlu) in Comparison to Two Different Formulations of Azelastine and Fluticasone Propionate Following Intranasal Administration in Healthy Chinese Volunteers.

Objective: Two studies (Study I and Study II) were conducted in healthy Chinese volunteers to confirm that there was no pharmacokinetic drug interaction between AZE and FLU in MP-AzeFlu. The secondary objective was to evaluate the pharmacokinetic parameters of MP-AzeFlu compared with the commercially available mono-components. Methods: Both studies were a randomized, open-label, three-period, six-sequence, single-dose cross-over trial (William's design) conducted at Beijing Hospital (Beijing, China) in September and October of 2019 in 30 healthy adult male and female volunteers. The natural log transformed parameters: AUC0-tlast, AUC0-∞ and Cmax were analyzed. Results: The comparison of PK parameters between MP-AzeFlu and Aze (commercially available) showed that the LS mean ratios (90% CI) values for, AUC0-tlast, AUC 0-∞ and Cmax were 100.29% (94.31-106.66%), 100.76% (94.60-107.32%) and 93.14% (81.47-106.48%). The comparison of PK parameters between MP-AzeFlu and Flu (commercially available) for the bioavailability evaluation showed that the LS mean ratios (90% CI) values for, AUC0-tlast, AUC 0-∞ and Cmax were 83.48% (69.81-99.82%), 100.19% (87.34-114.94%) and 81.91% (68.50-97.95%). Conclusion: The study results confirm that neither the FLU or the AZE component in the combination product (MP-AzeFlu), nor the existing qualitative and quantitative differences in the formulation between the currently marketed AZE and FLU mono-product, display significant potential to impact the systemic exposure of AZE or FLU in Chinese subjects.

Endothelial lipase: regulation and biological function.

Endothelial lipase is synthetized almost exclusively in endothelial cells and then fixed on the luminal surface of the endothelium by means of heparan sulphate proteoglycans. The enzyme is expressed in the endothelium of nearly all tissues and the degree of expression is higher in richly vascularized tissues than in the less vascularized ones. The endothelial lipase expression in tissues is upregulated by shear and cyclic stress, angiotensin II and hypertension. The plasma enzyme level is elevated by pro-inflammatory cytokines, in metabolic syndrome and obesity. Prolonged exercise reduces the plasma enzyme level in the rat. The activity of the enzyme is inhibited by: sphingomyelin, angiopoietin-like protein 3 and 4, and insulin. Endothelial lipase reduces the plasma high density lipoprotein concentration and changes its properties. The enzyme is considered to be the main regulator of the plasma high density lipoprotein concentration. The plasma endothelial lipase concentration is elevated in coronary atherosclerosis and it is inversely correlated with the plasma high density lipoprotein level. The enzyme is considered to exert mostly pro-atherogenic effects. Its action as triglyceride lipase is important in hypertriglyceridemia.

Wnt/ß-catenin Signaling Controls Maxillofacial Hyperostosis.

The roles of Wnt/ß-catenin signaling in regulating the morphology and microstructure of craniomaxillofacial (CMF) bones was explored using mice carrying a constitutively active form of ß-catenin in activating Dmp1-expressing cells (e.g., daßcatOt mice). By postnatal day 24, daßcatOt mice exhibited midfacial truncations coupled with maxillary and mandibular hyperostosis that progressively worsened with age. Mechanistic insights into the basis for the hyperostotic facial phenotype were gained through molecular and cellular analyses, which revealed that constitutively activated ß-catenin in Dmp1-expressing cells resulted in an increase in osteoblast number and an increased rate of mineral apposition. An increase in osteoblasts was accompanied by an increase in osteocytes, but they failed to mature. The resulting CMF bone matrix also had an abundance of osteoid, and in locations where compact lamellar bone typically forms, it was replaced by porous, woven bone. The hyperostotic facial phenotype was progressive. These findings identify for the first time a ligand-independent positive feedback loop whereby unrestrained Wnt/ß-catenin signaling results in a CMF phenotype of progressive hyperostosis combined with architecturally abnormal, poorly mineralized matrix that is reminiscent of craniotubular disorders in humans.

Effect of acute exercise on mRNA and protein expression of main components of the lipolytic complex in different skeletal muscle types in the rat.

Adipose triglyceride lipase (ATGL) hydrolyses the first bond of triacylglycerols. The activity of the enzyme is elevated by comparative gene identification 58 (CGI-58), and reduced by G0/G1 switch gene 2 (G0S2) protein. There are no data on the effect of acute exercise on the behavior of particular components of the lipolytic complex in different skeletal muscle types, therefore, the aim of the present study was to examine that topic. The experiments were carried out on four groups of male Wistar rats: 1) control 2) rats running on a treadmill at the speed of 18 m/min for 30 min, 3) at the speed of 18 m/min for 120 min, 4) for 30 min at the speed of 28 m/min. We found that each exercise bout induced numerous changes in the expression of mRNA and protein ATGL, hormone-sensitive lipase, CGI-58 and G0S2 in the investigated muscles. These changes, depended to a large extent on a muscle type. In general, the strongest pro-lipolytic response was observed in the soleus, followed by the red section of the gastrocnemius (RG). On the other hand, in the white section of the gastrocnemius protein expression of the components of the lipolytic complex was reduced in response to exercise. These changes were not accompanied by alterations in muscle triacylglycerol content, with the exception of a reduction observed in the RG following 2-hour run. We conclude that a single bout of exercise induces significant effect on the expression of components of the lipolytic complex in skeletal muscle, and that the magnitude of this effect depends on muscle oxidative capacity, as well as the duration and intensity of exercise.

Non-ischemic heart preconditioning.

Ischemic heart conditioning has been shown to protect the organ against ischemia/reperfusion injury. Animal studies have revealed that the heart can also be conditioned by non-ischemic procedures, namely physical exercise and tachycardia. Long and short term endurance training, sprint training, resistance or interval training and even one bout of exercise induce cardiac preconditioning, which is manifested by a reduction in post ischemia/reperfusion infarct size, ventricular arrhythmia and improved heart function. Several factors contribute to the exercise-induced heart preconditioning, among which the most important can be: increased activity of the anti-radical defense system, opioids, interleukin-6, nitric oxide, ATP dependent potassium channels, heat shock protein 72 and sphingosine-1-phosphate. A few studies have also shown that one bout of exercise in patients with stable angina increases tolerated workload. According to some data obtained in swine and dogs, stimulated tachycardia before ischemia/reperfusion reduces the infarct size. Future studies are needed to fully clarify the mechanisms responsible for exercise- or tachycardia-induced heart preconditioning against ischemia/reperfusion. It may lead to the development of new treatment modes of the disease.

Oral cancer radiotherapy affects enamel microhardness and associated indentation pattern morphology.

OBJECTIVES: The aim of this study is to determine the effects of in vitro and in vivo high-dose radiotherapy on microhardness and associated indentation pattern morphology of enamel. MATERIALS AND METHODS: The inner, middle, and outer microhardness of enamel was evaluated using three experimental groups: control (non-radiated); in vitro irradiated; in vivo irradiated. In vitro specimens were exposed to simulated radiotherapy, and in vivo specimens were extracted teeth from oral cancer patients previously treated with radiotherapy. Indentations were measured via SEM images to calculate microhardness values and to assess the mechanomorphological properties of enamel before and after radiotherapy. RESULTS: Middle and outer regions of enamel demonstrated a significant decrease in microhardness after in vitro and in vivo irradiation compared to the control group (p < 0.05). Two indentation patterns were observed: pattern A-presence of microcracks around indent periphery, which represents local dissipation of deformation energy; pattern B-clean, sharp indents. The percentage of clean microindentation patterns, compared to controls, was significantly higher following in vitro and in vivo irradiation in all enamel regions. The highest percentage of clean microindentations (65%) was observed in the in vivo irradiated group in the inner region of enamel near the dentin-enamel junction. CONCLUSIONS: For the first time, this study shows that in vitro and in vivo irradiation alters enamel microhardness. Likewise, the indentation pattern differences suggest that enamel may become more brittle following in vitro and in vivo irradiation. CLINICAL RELEVANCE: The mechanomorphological property changes of enamel following radiation may be a contributory component of pathologic enamel delamination following oral cancer radiotherapy.

The skeletal and heart muscle triacylglycerol lipolysis revisited.

For 40 years, the enzyme hormone sensitive lipase was considered to hydrolyze the first ester bond of the triacylglycerol moiety and thus initiate hydrolysis. However, 12 years ago a new lipolytic enzyme, termed adipose triglyceride lipase was discovered. It was further shown that the process of lipolysis of triacylglycerol to diacylglycerol and fatty acid is initiated by adipose triglyceride lipase and not by hormone sensitive lipase, responsible for hydrolysis of diacylglycerol to monoacyglycerol and fatty acid. Adipose triglyceride lipase is present in all types of cells containing neutral fat. The enzyme is activated by a protein called comparative gene identification-58 and inhibited by a protein called G0/G1 switch protein 2. It has also been discovered that perilipins, the main proteins coating lipid droplets in the cells, are involved in the process of triacylglycerol lipolysis. Five perilipins (1-5) were identified, however, up to now their role has been poorly assessed. In skeletal muscles, exercise and training affect the mRNA expression and protein content of adipose triglyceride lipase, comparative gene identification-58, G0/G1 switch protein 2, perilipin 2 and 5. The effect of exercise/training depends on exercise intensity and type of muscle fiber. An interaction between comparative gene identification-58 and adipose triglyceride lipase seems to be responsible for the enzyme activation during contractile activity. Adipose triglyceride lipase is also responsible for the activation of the first step of triacylglycerol lipolysis in the heart. There is substantial evidence that cardiac triacylglycerol metabolism affects the function of the heart. ATGL gene mutations leads to the development of neutral lipid storage diseases.

Effect of tachycardia on mRNA andf protein expression of the principal components of the lipolytic system in the rat's heart ventricles.

There is a convincing piece of evidence showing that most of free fatty acids (FFA) entering cardiomyocytes are first esterified into triacylglycerols (TG) before being oxidized or used for synthesis of complex lipids. The enzyme adipose triglyceride lipase (ATGL) initiates lipolysis of TG by hydrolyzing the first ester bond of the compound. As a result, free fatty acid and diacylglycerol (DG) are released in that process. Finally, DG may be further hydrolyzed by the enzyme hormone sensitive lipase (HSL). The aim of the present study was to examine effect of tachycardia on mRNA and protein expression of ATGL, CGI-58 (an activator of ATGL), G0S2 (an inhibitor of ATGL) and HSL in the left and right ventricle of the rat. The experiments were carried out on male Wistar rats, 240 - 260 grams of body weight. After anesthesia, two electrodes were inserted in the right jugular vein and connected to SC-04 stimulator. The rats were randomly allocated into one of the three groups, namely: control, 30 min and 60 min of the heart stimulation at the rate of 600 times/min. The expressions of ATGL, CGI-58, G0S2 and HSL were evaluated at both gene and protein levels using real-time PCR and Western Blot analysis, respectively. Both 30 and 60 min stimulation reduced ATGL, HSL, CGI-58 and G0S2 mRNA content in the left ventricle. The stimulation had only insignificant impact on ATGL, HSL and CGI-58 transcript levels in the right ventricle. Interestingly, Western Blot analysis showed that the stimulation, regardless of the time, reduced the ATGL and G0S2 protein expression, but did not change the CGI-58 and HSL expression in the left ventricle. Furthermore, in the right ventricle, the protein expressions of ATGL, HSL and G0S2 decreased after stimulation. In conclusion, the obtained results clearly show that tachycardia affects both mRNA and protein expression of particular compounds of the TG lipolytic system in the heart ventricles. Additionally, there are marked differences in the expressions of the examined compounds between the ventricles.

Effect of tachycardia on lipid metabolism and expression of fatty acid transporters in heart ventricles of the rat.

Tachycardia increases oxidation of the plasma-borne long chain fatty acids in the heart. The aim of the present study was to examine effect of tachycardia on: 1) the total level of free fatty acids, diacylglycerols, triacylglycerols and phospholipids in both heart ventricles; 2) (14)C-palmitate incorporation in the lipid fractions; 3) expression of fatty acid and glucose transporters in the ventricles. Tachycardia was induced in anesthetized rats by electrical atrial pacing at the rate of 600/min. Samples of the left (LV) and right (RV) ventricle were taken after 30 and 60 min pacing. The level free fatty acids, diacylglycerols, triacylglycerols and phospholipids was determined by means of gas-liquid chromatography and (14)C-palmitate incorporation by liquid scintillation counting, respectively. Expression of fatty acid- and glucose-transporters was determined using Western blot technique. In LV, 30min pacing increased the content of diacylglycerols whereas the content of other lipids remained stable. After 60 min of pacing the levels of the examined lipid fractions did not differ from the respective control values. In RV, the content of diacylglycerols and triacylglycerols was reduced both after 30 and 60 min pacing. Tachycardia also affected incorporation of (14)C-palmitate in lipid fractions of goth ventricles. 30 min pacing up-regulated plasmalemmal expression of FAT/CD36 (fatty acid translocase) in both ventricles and reduced its microsomal expression in LV. After 60 min pacing they did not differ from the respective control values. Plasmalemmal expression of FATP-1 (fatty acid transport protein 1) increased and its microsomal expression decreased in RV after 30 min pacing. After 60 min pacing the plasmalemmal FATP-1 expression remained elevated whereas the microsomal expression did not differ from the control value. Pacing did not affect or expression of FABPpm (plasma membrane associated fatty acid binding protein) in either plasma membranes and microsomal compartments. Thirty min pacing increased plasmalemmal and reduced microsomal expression of GLUT-4 (glucotransporter 4) in both ventricles. It increased plasmalemmal expression of GLUT-1 (glucotransporter 1) in RV. It returned to normal after 60 min pacing. It is concluded that tachycardia induces numerous changes in metabolism of myocardial lipids as well as expression of fatty acid and glucose transporters in both heart ventricles.

Effect of atrial pacing on the level of bioactive sphingolipids in the heart ventricles of the rat.

Bioactive sphingolipids play important role in regulation of the function of the cardiomyocytes. There are no data available on metabolism of the sphingolipids in the heart under increased work-load produced by tachycardia. The aim of the present study was to examine effect of tachycardia on the level of the principal bioactive sphingolipids in the left and right ventricles. The experiments were carried out on male Wistar rats. After anesthesia, two electrodes were administered into the right common jugular vein so that their tips were placed at the vein's aperture. The resting heart rate was 355 ± 24/min and the rate of stimulation was 600/min. EKG was continuously monitored. The stimulation time was 30 and 60 min. Thereafter, blood from the abdominal aorta and samples of the left and right ventricle were taken. The following bioactive sphingolipids were quantified by means of high performance liquid chromatography: sphinganine, ceramide, sphingosine, sphingosine-1-phosphate and sphinganine-1-phosphate. In the left ventricle, 30 and 60 min tachycardia elevated the level of sphingosine, reduced the level of sphingosine-1-phosphate and sphinganine-1-phosphate. The level of ceramide was reduced only after 60 min. In the right ventricle, 60 min pacing resulted in elevation in the level of sphingosine and sphinganine and reduction in the level of other compounds studied. It is concluded that tachycardia induces changes in metabolism of bioactive sphingolipids in each ventricle. The changes may affect cardiomyocyte functions. Also, differences in sphingolipid metabolism between both ventricles are reported.

Radiotherapy effect on nano-mechanical properties and chemical composition of enamel and dentine.

OBJECTIVE: To understand radiotherapy-induced dental lesions characterized by enamel loss or delamination near the dentine-enamel junction (DEJ), this study evaluated enamel and dentine nano-mechanical properties and chemical composition before and after simulated oral cancer radiotherapy. DESIGN: Sections from seven non-carious third molars were exposed to 2 Gy fractions, 5 days/week for 7 weeks for a total of 70 Gy. Nanoindentation was used to evaluate Young's modulus, while Raman microspectroscopy was used to measure protein/mineral ratios, carbonate/phosphate ratios, and phosphate peak width. All measures were completed prior to and following radiation at the same four buccal and lingual sites 500 and 30 µm from the DEJ in enamel and dentine (E-500, E-30, D-30 and D-500). RESULTS: The elastic modulus of enamel and dentine was significantly increased (P ≤ 0.05) following radiation. Based on Raman spectroscopic analysis, there was a significant decrease in the protein to mineral ratio (2931/430 cm(-1)) following radiation at all sites tested except at D-500, while the carbonate to phosphate ratio (1070/960 cm(-1)) increased at E-30 and decreased at D-500. Finally, phosphate peak width as measured by FWHM at 960 cm(-1) significantly decreased at both D-30 and D-500 following radiation. CONCLUSIONS: Simulated radiotherapy produced an increase in the stiffness of enamel and dentine near the DEJ. Increased stiffness is speculated to be the result of the radiation-induced decrease in the protein content, with the percent reduction much greater in the enamel sites. Such changes in mechanical properties and chemical composition could potentially contribute to DEJ biomechanical failure leading to enamel delamination that occurs post-radiotherapy. However, other analyses are required for a better understanding of radiotherapy-induced effects on tooth structure to improve preventive and restorative treatments for oral cancer patients.

Type IV collagen is a novel DEJ biomarker that is reduced by radiotherapy.

The dental basement membrane (BM) is composed of collagen types IV, VI, VII, and XVII, fibronectin, and laminin and plays an inductive role in epithelial-mesenchymal interactions during tooth development. The BM is degraded and removed during later-stage tooth morphogenesis; however, its original position defines the location of the dentin-enamel junction (DEJ) in mature teeth. We recently demonstrated that type VII collagen is a novel component of the inner enamel organic matrix layer contiguous with the DEJ. Since it is frequently co-expressed with and forms functional complexes with type VII collagen, we hypothesized that type IV collagen should also be localized to the DEJ in mature human teeth. To identify collagen IV, we first evaluated defect-free erupted teeth from various donors. To investigate a possible stabilizing role, we also evaluated extracted teeth exposed to high-dose radiotherapy--teeth that manifest post-radiotherapy DEJ instability. We now show that type IV collagen is a component within the morphological DEJ of posterior and anterior teeth from individuals aged 18 to 80 yr. Confocal microscopy revealed that immunostained type IV collagen was restricted to the 5- to 10-µm-wide optical DEJ, while collagenase treatment or previous in vivo tooth-level exposure to > 60 Gray irradiation severely reduced immunoreactivity. This assignment was confirmed by Western blotting with whole-tooth crown and enamel extracts. Without reduction, type IV collagen contained macromolecular α-chains of 225 and 250 kDa. Compositionally, our results identify type IV collagen as the first macromolecular biomarker of the morphological DEJ of mature teeth. Given its network structure and propensity to stabilize the dermal-epidermal junction, we propose that a collagen-IV-enriched DEJ may, in part, explain its well-known fracture toughness, crack propagation resistance, and stability. In contrast, loss of type IV collagen may represent a biochemical rationale for the DEJ instability observed following oral cancer radiotherapy.

Extracts of irradiated mature human tooth crowns contain MMP-20 protein and activity.

OBJECTIVES: We recently demonstrated a significant correlation between enamel delamination and tooth-level radiation dose in oral cancer patients. Since radiation can induce the synthesis and activation of matrix metalloproteinases, we hypothesized that irradiated teeth may contain active matrix metalloproteinases. MATERIALS AND METHODS: Extracted teeth from oral cancer patients treated with radiotherapy and from healthy subjects were compared. Extracted mature third molars from healthy subjects were irradiated in vitro and/or incubated for 0-6 months at 37°C. All teeth were then pulverized, extracted, and extracts subjected to proteomic and enzymatic analyses. RESULTS: Screening of irradiated crown extracts using mass spectrometry identified MMP-20 (enamelysin) which is expressed developmentally in dentine and enamel but believed to be removed prior to tooth eruption. MMP-20 was composed of catalytically active forms at Mr=43, 41, 24 and 22kDa and was immunolocalized predominantly to the morphological dentine enamel junction. The proportion of different sized MMP-20 forms changed with incubation and irradiation. While the pattern was not altered directly by irradiation of healthy teeth with 70Gy, subsequent incubation at 37°C for 3-6 months with or without prior irradiation caused the proportion of Mr=24-22kDa MMP-20 bands to increase dramatically. Extracts of teeth from oral cancer patients who received >70Gy radiation also contained relatively more 24 and 22kDa MMP-20 than those of healthy age-related teeth. CONCLUSION: MMP-20 is a radiation-resistant component of mature tooth crowns enriched in the dentine-enamel. We speculate that MMP-20 catalyzed degradation of organic matrix at this site could lead to enamel delamination associated with oral cancer radiotherapy.

Evaluation of partial area under the concentration time curve to estimate midazolam apparent oral clearance for cytochrome P450 3A phenotyping.

BACKGROUND: Midazolam apparent oral clearance (CLORAL) is used to estimate intestinal and hepatic cytochrome P450 (CYP) 3A activity. A limited sampling approach was performed to access a midazolam partial area under the concentration time curve (AUC) to estimate CLORAL. METHODS: Midazolam plasma concentrations from healthy adults were obtained during CYP3A baseline (n=116), inhibition (n=75), and induction or activation (n=66) from seven published studies. Observed CLORAL and partial AUCs of AUC0-2, AUC0-4, AUC0-6, AUC1-2, AUC1-4, AUC2-4, and AUC2-6 were determined by noncompartmental analysis. Subject data were randomly divided into a training set and a validation set. Linear regression equations, derived from partial AUCs, were developed from training set data. Predicted CLORAL was determined from these equations from validation set data. Preset criterion was a coefficient of determination (r2) greater than or equal to 0.9. Bias and precision were evaluated by relative percent mean prediction error (%MPE) and relative percent mean absolute error (%MAE). RESULTS: During CYP3A baseline conditions, all of the evaluated CLORAL equations had unacceptable r2 (range: 0.34-0.86). During CYP3A inhibition, all of the evaluated CLORAL equations had unacceptable %MAE. Acceptable r2, %MPE, and %MAE were observed during CYP3A induction/activation with AUC0-4 (r2=0.99, %MPE=3.9, %MAE=12.5) and AUC1-4 (r2=0.99, %MPE=6%, %MAE=11.1%). The same equations also predicted the extent of CYP3A induction as a lack of equivalence was observed with AUC0-4 and AUC1-4. CONCLUSIONS: Midazolam partial AUCs were unable to estimate CYP3A activity during the evaluated baseline and inhibitory conditions. Midazolam CLORAL utilizing a partial AUC0-4 and AUC1-4 was able to estimate CYP3A induction with rifampin and Ginkgo biloba extract.

Liver X receptor agonist T0901317 enhanced peroxisome proliferator-activated receptor-delta expression and fatty acid oxidation in rat skeletal muscle.

Liver X receptors (LXR) have been characterized as key transcriptional regulators of hepatic lipid and carbohydrate metabolism. LXR are expressed also in skeletal muscle, however, their role in this tissue is poorly investigated and the vast majority of available data comes from studies on cultured myotubes. Therefore, we aimed to examine effects of in vivo LXR activation on muscle lipid metabolism. The experiments were performed on male Wistar rats fed on a standard rodent chow. The animals were divided into two groups (n=10) receiving either LXR activator (T0901317, 10 mg/kg/day) or vehicle for one week. Samples of the soleus as well as red and white sections of the gastrocnemius muscle were excised. T0901317 increased muscle expression of peroxisome proliferator-activated receptor-δ and its target genes involved in fatty acid uptake and oxidation. In addition, LXR agonist enhanced palmitate oxidation (by 55%) in isolated soleus muscle. However, palmitate incorporation into triacylglycerol was decreased (by 38%), which was associated with reduced diacylglycerol acyltransferase expression (by 66%). Despite markedly increased plasma lipid concentration upon T0901317 treatment, muscle triacylglycerol level was elevated only in the red section of the gastrocnemius muscle. We conclude that T0901317 enhances muscle fatty acid oxidation, which prevents overt accumulation of intramuscular lipids that could be expected considering T0901317-induced hyperlipidemia.

Further Evidence of Contrasting Phenotypes Caused by Reciprocal Deletions and Duplications: Duplication of NSD1 Causes Growth Retardation and Microcephaly.

Microduplications of the Sotos syndrome region containing NSD1 on 5q35 have recently been proposed to cause a syndrome of microcephaly, short stature and developmental delay. To further characterize this emerging syndrome, we report the clinical details of 12 individuals from 8 families found to have interstitial duplications involving NSD1, ranging in size from 370 kb to 3.7 Mb. All individuals are microcephalic, and height and childhood weight range from below average to severely restricted. Mild-to-moderate learning disabilities and/or developmental delay are present in all individuals, including carrier family members of probands; dysmorphic features and digital anomalies are present in a majority. Craniosynostosis is present in the individual with the largest duplication, though the duplication does not include MSX2, mutations of which can cause craniosynostosis, on 5q35.2. A comparison of the smallest duplication in our cohort that includes the entire NSD1 gene to the individual with the largest duplication that only partially overlaps NSD1 suggests that whole-gene duplication of NSD1 in and of itself may be sufficient to cause the abnormal growth parameters seen in these patients. NSD1 duplications may therefore be added to a growing list of copy number variations for which deletion and duplication of specific genes have contrasting effects on body development.

Fiber specific changes in sphingolipid metabolism in skeletal muscles of hyperthyroid rats.

Thyroid hormones (T3, T4) are well known modulators of different cellular signals including the sphingomyelin pathway. However, studies regarding downstream effects of T3 on sphingolipid metabolism in skeletal muscle are scarce. In the present work we sought to investigate the effects of hyperthyroidism on the activity of the key enzymes of ceramide metabolism as well as the content of fundamental sphingolipids. Based on fiber/metabolic differences, we chose three different skeletal muscles, with diverse fiber compositions: soleus (slow-twitch oxidative), red (fast-twitch oxidative-glycolytic) and white (fast-twitch glycolytic) section of gastrocnemius. We demonstrated that T3 induced accumulation of sphinganine, ceramide, sphingosine, as well as sphingomyelin, mostly in soleus and in red, but not white section of gastrocnemius. Concomitantly, the activity of serine palmitoyltransferase and acid/neutral ceramidase was increased in more oxidative muscles. In conclusion, hyperthyroidism induced fiber specific changes in the content of sphingolipids that were relatively more related to de novo synthesis of ceramide rather than to its generation via hydrolysis of sphingomyelin.

Change in blood gelsolin concentration in response to physical exercise.

Plasma gelsolin (pGSN) produced by muscle is an abundant protein of extracellular fluids capable of severing actin filaments and eliminating actin from the circulation. Additionally, pGSN modulates the cellular effects of some bioactive lipids. In this study we test the hypothesis that hormonal and metabolic adaptations to exercise are associated with changes in gelsolin concentration in blood. Plasma samples were collected from twenty healthy males recruited from untrained (UT, n=10) and endurance trained (ET, n=10) groups that performed 30-60 minutes of exercise on a cycloergometer at a workload corresponding to 70% of VO2max. Gelsolin concentration was determined by quantitative Western blot analysis with an anti-human gelsolin antibody. The gelsolin concentration in UT and ET subjects before starting exercise ranged from 104 to 330 and 163 to 337 µg · ml(-1) respectively. After 30 minutes of exercise we observed a significant decrease of plasma gelsolin in the UT group (p<0.05) while the gelsolin concentration in the ET group rose on average from 244 to 271 µg · ml(-1). However, this increase did not reach statistical significance. Endurance training might increase the ability of muscle tissue to express plasma gelsolin as part of an adaptive mechanism.

Fatty acid transporters involved in the palmitate and oleate induced insulin resistance in primary rat hepatocytes.

AIMS: To determine the presence and possible involvement of FAT/CD36, FABPpm and FATP-2, transporters in (i) fatty acids movement across plasma membrane and (ii) an induction of insulin resistance by palmitic (PA) and oleic (OA) fatty acids in primary hepatocytes. METHODS: Primary hepatocytes were treated with either PA and OA or combination of activators (AICAR, Insulin) or inhibitors (SSO, phloretin) of FA transport. Expression of FA and glucose transporters as well as insulin signalling proteins was determined using Western blot analyses. Palmitate and glucose transport was measured using radioactive isotopes. Intracellular lipid content [ceramide, diacylglycerols (DG) and triacylglycerols] and FA composition were estimated by GLC. RESULTS: In primary hepatocytes, adding phloretin diminished insulin, and AICAR stimulated palmitate transport. Both PA and OA fatty acids induced the protein expression of FAT/CD36 and FATP-2 with concomitant: (i) reduction in GLUT-2 protein content, (ii) inhibition of insulin-stimulated glucose uptake, (iii) reduction in insulin-stimulated activation of AKT and GSK, (iv) accumulation of either DG (PA and OA) or ceramide (only PA). CONCLUSIONS: FA transport into hepatocytes is, at least in part, protein-mediated process, and both PA and OA induce the protein expression of FAT/CD36 and FATP-2. Both saturated (PA) and unsaturated (OA) fatty acids induce insulin resistance in primary hepatocytes, associated with the accumulation of DG and/or ceramide.

Rate of onset of inhibition of gut-wall and hepatic CYP3A by clarithromycin.

AIMS: To determine the extent and time-course of hepatic and intestinal cytochrome P450 3A (CYP3A) inactivation due to the mechanism-based inhibitor clarithromycin. METHODS: Intestinal and hepatic CYP3A inhibition was examined in 12 healthy volunteers following the administration of single and multiple doses of oral clarithromycin (500 mg). Intestinal biopsies were obtained under intravenous midazolam sedation at baseline and after the first dose, on days 2-4, and on days 6-8 of the clarithromycin treatment. The formation of 1'-hydroxymidazolam in biopsy tissue and the serum 1'-hydroxymidazolam:midazolam ratio were indicators of intestinal and hepatic CYP3A activity, respectively. RESULTS: Intestinal CYP3A activity decreased by 64 % (p = 0.0029) following the first dose of clarithromycin, but hepatic CYP3A activity did not significantly decrease. Repeated dosing of clarithromycin caused a significant decrease in hepatic CYP3A activity (p = 0.005), while intestinal activity showed little further decline. The CYP3A5 or CYP3A4*1B genotype were unable to account for inter-individual variability in CYP3A activity. CONCLUSIONS: Following the administration of clarithromycin, the onset of hepatic CYP3A inactivation is delayed compared to that of intestinal CYP3A. The time-course of drug-drug interactions due to clarithromycin will vary with the relative contribution of intestinal and hepatic CYP3A to the clearance and bioavailability of a victim substrate.