mTOR inhibition improves fibroblast growth factor receptor targeting in hepatocellular carcinoma.

BACKGROUND: Systemic therapy has proven only marginal effects in hepatocellular carcinoma (HCC) so far. The aim of this study was to evaluate the effect of targeting fibroblast growth factor receptor (FGFR) on tumour and stromal cells in HCC models. METHODS: Human and murine HCC cells, endothelial cells (ECs), vascular smooth muscle cells (VSMCs), hepatic stellate cells (HSCs), human HCC samples, FGFR inhibitor BGJ398 and mammalian target of rapamycin (mTOR) inhibitor rapamycin were used. Effects on growth, motility, signalling and angiogenic markers were determined. In vivo subcutaneous and syngeneic orthotopic tumour models were used. RESULTS: In tumour cells and ECs, targeting FGFR showed significant inhibitory effects on signalling and motility. Minor effects of FGFR inhibition were observed on VSMCs and HSCs, which were significantly enhanced by combining FGFR and mTOR blockade. In vivo daily (5 mg kg(-1)) treatment with BGJ398 led to a significant growth inhibition in subcutaneous tumour models, but only a combination of FGFR and mTOR blockade impaired tumour growth in the orthotopic model. This was paralleled by reduced tumour cell proliferation, vascularisation, pericytes and increased apoptosis. CONCLUSIONS: Targeting FGFR with BGJ398 affects tumour cells and ECs, whereas only a combination with mTOR inhibition impairs recruitment of VSMCs and HSCs. Therefore, this study provides evidence for combined FGFR/mTOR inhibition in HCC.

Generation of 1,2-azaboretidines via reduction of ADC borane adducts.

Reaction of the acyclic (diamino)carbene (ADC) :C(NiPr2)2 (1) with different dihaloboranes of the type RBX2 (R = Mes, Dur; X = Cl, Br) smoothly afforded a novel class of ADC-stabilized borane adducts. For MesBBr2 however, the reaction did not stop at the adduct level, but an uncommon rearrangement process occurred, which eventually resulted in the formation of a 5-membered boracycle after elimination of mesitylene. Chemical reduction of the ADC borane adducts by KC8 selectively yielded air stable 1,2-azaboretidines. Detailed DFT studies suggest a reduction mechanism involving a highly reactive borylene intermediate, which is converted into the boracycles via a rearrangement/C-H activation sequence.

Influence of a reduced gravity on the volume fraction of a monolayer of spherical grains.

Centrifuge force is used to study granular materials in low gravity conditions. We consider a monolayer of noncohesive spherical grains placed on a plate. Reduced gravity conditions can be simulated in the plane by tilting or by rotating the plate. We compare both approaches experimentally. The volume fraction is found to increase with the apparent gravity and saturates. A model based on the exponential distribution of the Voronoi cell areas has been built and is in excellent agreement with the experimental data by extrapolating the fits of the data. Moreover, numerical simulations exhibit that more arches can be maintained at low apparent gravities than at high.

Precursors to avalanches in a granular monolayer.

We investigate the stability of a granular monolayer composed of spherical grains on an inclined plate. When the tilt angle alpha increases, some reorganizations are observed throughout the pile. The packing fraction rho of the packing evolves by successive jumps. Those discontinuous events precede the collapse of the pile at a critical angle alphac. The occurrence of precursors before avalanches is modeled by stop-and-go motions of blocks due to the competition between sliding friction and the Janssen effect [J. Durand, (Springer-Verlag, New York, 2000)].

Establishment of a novel in vitro system for studying the interaction of xenobiotic metabolism of liver and intestinal microflora.

We developed a new two-chamber system for the coculture of hepatocytes and fecal microflora under aerobic and anaerobic conditions, respectively, to investigate the sequential metabolism of chemicals by the liver and microflora in vitro. The culture device consisted of two chambers separated by a permeable polycarbonate membrane. In the aerobic compartment, hepatocytes were cultivated as a monolayer on the membrane and in the anaerobic compartment fecal microflora as a suspension. To characterize the metabolic capacity of the microflora and hepatocytes, various marker enzymes were studied. Azoreductase, nitroductase, beta-glucuronidase, beta-glucosidase and sulphatase were tested in the microflora of the feces from three volunteers who had had significantly different eating habits for years (daily meat, mixed diet, vegetarian). The microflora exhibited significant activities and the various enzymes differed only moderately in the samples from the three volunteers. For rat hepatocytes the activities of various cytochrome P450 forms and conjugating enzymes served as markers. The enzyme activities were tested in the coculture system during a 4-h culture period intended for the test protocol. Deethylation of ethoxycoumarin and 2alpha-, 6beta- and 16alpha-hydroxylation of testosterone decreased by about 30%, 25%, 40% and 20%, respectively, while there was no loss of glucuronidation and sulphonation of 3-OH-benzo(a)pyrene nor of glutathione conjugation of 1-chloro-2,4-dinitrobenzene during the 4-h culture period. The activities of the tested hepatic phase I and II enzymes were not changed after coculture of the hepatocytes with the microflora for 4 h. The applicability of the in vitro system for studying the metabolic interaction of liver and microflora was demonstrated using 7-ethoxycoumarin and the developmental drug EMD 57033, a thiadiazinon derivative from Merck KGaA, as model compounds. Both compounds were oxidized and conjugated by liver cells. In the coculture of hepatocytes and fecal microflora the resulting glucuronides and sulphoconjugates were split by hydrolytic enzymes of the intestinal microflora.

Duration, compression, and the aided loudness discomfort level.

OBJECTIVE: The purpose of this investigation is to determine how the unaided and aided loudness discomfort level (LDL) varies with the duration of the input signal and whether the electroacoustic characteristics of compression circuits affect this relationship in a manner that may alter the listener's dynamic range for short duration sounds. DESIGN: Ten hearing-impaired and 20 normal-hearing listeners participated. LDLs were determined for noise bursts of durations ranging in six steps from 32 to 1024 msec, using a two-alternative, forced-choice adaptive tracking procedure in which input level varied until LDL was achieved. LDLs were also obtained for continuous discourse, using a clinical procedure. Subjects were also given the opportunity to self adjust maximum output SPL to their LDL using either output limiting or volume controls in response to fixed 90 dB SPL noise bursts. Testing was conducted unaided and with hearing aids representing two analog (output compression limiting, wide dynamic range compression) and four digital compression circuits. Primary circuit contrasts included compression threshold, compression ratio, attack time and the presence or absence of unity gain at high levels. RESULTS: For the unaided condition, both normal-hearing and hearing-impaired subjects showed increasing LDLs with decreasing signal duration. Under aided conditions, circuits with compression thresholds of 45 to 50 dB SPL and compression ratios of 2:1 produced LDL functions that were similar in slope to the impaired listener's unaided functions. Slopes were steeper when the attack time was slow (128 msec) than when it was fast (2 msec). Circuits with compression ratios of 8:1 produced flat LDL duration functions (i.e., a loss of duration-dependent effects). Similar duration-dependent LDL effects were also observed when subjects adjusted their own hearing aid output characteristics in response to 90 dB noise bursts. CONCLUSION: For the unaided condition, results suggest that normal-hearing and hearing-impaired listeners can tolerate short duration sounds at higher levels than long duration sounds, a finding that has implications for hearing aid design. Circuits that preserve the relationship between duration and LDL should allow brief phonemes to be presented at higher levels without discomfort than circuits that do not, possibly resulting in greater audibility or speech recognition. Current results suggest that circuits with low compression thresholds, low compression ratios, and slow attack times might accomplish this objective better than circuits with high compression thresholds, high compression ratios and fast attack times.

Cloning of the multipartite promoter of the sodium-calcium exchanger gene NCX1 and characterization of its activity in vascular smooth muscle cells.

The sodium-calcium exchange activity is mediated by proteins encoded in a small gene family, of which the gene NCX1 is ubiquitously expressed in mammalian tissues. In this study, the multipartite promoter of this gene was analyzed in the human and rat genomes by means of DNA cloning, reverse transcriptase-polymerase chain reaction, and transient transfection of fusion constructs with the firefly luciferase gene into cultured rat aortic smooth muscle cells. The gene-proximal promoter, located 30 kilobase pairs (kb) away from the first coding exon 2, has features of a GC-rich housekeeping promoter and is apparently always active; in specific tissues, however, it is augmented by one or two additional promoters, located either within 1.5 kb upstream of it, or 35 kb upstream. The gene proximal promoter shows the highest activity in aortic smooth muscle cells. In mammalian species transcripts from all three promoters undergo splicing via an intermediate, containing two noncoding exons, of which the downstream one is normally not present in the terminal splicing product.

Magnetic resonance imaging in sheehan's syndrome: case report and literature review of imaging studies.

OBJECTIVE: To present the first documentation of pituitary atrophy on magnetic resonance imaging (MRI) in a patient with Sheehan's syndrome and review the published radiologic findings in this syndrome. METHODS: We describe the clinical and laboratory findings in a young woman with Sheehan's syndrome, provide the MRI results, and discuss the previously published radiologic studies of this syndrome. RESULTS: A 31-year-old woman, who was examined 11 months after severe postpartum hemorrhage, had clinical and biochemical findings consistent with Sheehan's syndrome (loss of axillary hair, amenorrhea, and impaired pituitary reserve for luteinizing hormone, follicle-stimulating hormone, thyrotropin, growth hormone, and corticotropin). MRI showed a partially empty sella in conjunction with invagination of the optic chiasm anteriorly into the pituitary fossa. CONCLUSION: Because of its greater spatial resolution, higher signal-to-noise ratio, and potential for multiplanar images, MRI can provide more precise and detailed findings than other radiologic studies and can facilitate diagnosis of Sheehan's syndrome.

Mouse liver nicotinamide N-methyltransferase pharmacogenetics: biochemical properties and variation in activity among inbred strains.

Nicotinamide N-methyltransferase (NNMT) catalyses the N-methylation of nicotinamide and other pyridines. Human liver NNMT activity shows large individual variations and a bimodal frequency distribution, raising the possibility that this activity, like those of many other methyltransferase enzymes, might be regulated by a genetic polymorphism. In an attempt to develop an experimental animal model for pharmacogenetic studies of NNMT, we determined optimal conditions for the measurement of hepatic NNMT activity in C57BL/6J mice. Mouse liver NNMT was a cytoplasmic enzyme with a pH optimum of 7.4 and apparent Km values for nicotinamide and S-adenosyl-L-methionine, cosubstrates for the reaction, of 370 and 6.5 microM, respectively. These properties were very similar to those of human liver NNMT, as was the relative sensitivity of the mouse liver enzyme to a series of methyltransferase inhibitors. Hepatic NNMT activity was then measured in tissue from male mice of 10 inbred strains. Average levels of NNMT activity in these strains varied by up to 14-fold and ranged from 1.13 +/- 0.18 U per mg protein (mean +/- SEM, n = 6) for C3H/HeJ mice to 16.0 +/- 1.16 U per mg protein in C57BR/cdJ animals. Average hepatic NNMT activities in female mice of six strains in which both sexes were studied varied from five-fold higher than those in males for "low activity' strains, to not significantly different for "high activity' strains. A series of properties of NNMT was then compared in hepatic cytosol from male mice of three different strains - one with "low' (C3H/HeJ), one with "intermediate' (DBA/2J), and one with "high' (C57BL/6J) hepatic NNMT activity. There were no striking differences among these three strains in hepatic NNMT pH optimum, substrate kinetics, IC50 values for inhibitors, thermal stability or behavior during ion exchange chromatography. The existence of large strain and gender-dependent variation in hepatic NNMT activity will make it possible to use inbred mice for studies of the role of inheritance and gender in the regulation of NNMT activity in this species, as well as for studies of the potential pharmacological and toxicological consequences of variation in this important drug-metabolizing enzyme activity.

Prospective evaluation of a nonradiographic device for determination of endotracheal tube position in children.

A new noninvasive, nonradiographic endotracheal tube (ETT) position detection system (ETT-PDS) for guidance of ETT positioning was evaluated in pediatric ICU patients. The system includes an ETT with a metallic element embedded at a defined distance from the ETT tip, and a portable locator instrument which detects transcutaneously the position of the metallic element. The contribution of ETT-PDS to accuracy of ETT positioning after intubation and before chest radiographs was evaluated in 92 critically ill children. The ETT malposition rates observed on the postintubation chest radiographs were 39.1% after positioning guided by clinical assessment alone, and 19.6% after positioning guided by clinical assessment plus the ETT-PDS (p less than 0.5). This reduction in malnutrition rate could not be demonstrated when the ETT-PDS was used to guide routine ETT positioning performed before morning chest radiographs.