Identification of high-affinity binding sites for the insulin sensitizer rosiglitazone (BRL-49653) in rodent and human adipocytes using a radioiodinated ligand for peroxisomal proliferator-activated receptor gamma

A radioiodinated ligand, [125I]SB-236636 [(S)-(-)3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]3-[125I]i odo phenyl]2-ethoxy propanoic acid], which is specific for the gamma isoform of the peroxisomal proliferator activated receptor (PPARgamma), was developed. [125I]SB-236636 binds with high affinity to full-length human recombinant PPARgamma1 and to a GST (glutathione S-transferase) fusion protein containing the ligand binding domain of human PPARgamma1 (KD = 70 nM). Using this ligand, we characterized binding sites in adipose-derived cells from rat, mouse and humans. In competition experiments, rosiglitazone (BRL-49653), a potent antihyperglycemic agent, binds with high affinity to sites in intact adipocytes (IC50 = 12, 4 and 9 nM for rat, 3T3-L1 and human adipocytes, respectively). Binding affinities (IC50) of other thiazolidinediones for the ligand binding domain of PPARgamma1 were comparable with those determined in adipocytes and reflected the rank order of potencies of these agents as stimulants of glucose transport in 3T3-L1 adipocytes and antihyperglycemic agents in vivo: rosiglitazone > pioglitazone > troglitazone. Competition of [125I]SB-236636 binding was stereoselective in that the IC50 value of SB-219994, the (S)-enantiomer of an alpha-trifluoroethoxy propanoic acid insulin sensitizer, was 770-fold lower than that of SB-219993 [(R)-enantiomer] at recombinant human PPARgamma1. The higher binding affinity of SB-219994 also was evident in intact adipocytes and reflected its 100-fold greater potency as an antidiabetic agent. The results strongly suggest that the high-affinity binding site for [125I]SB-236636 in intact adipocytes is PPARgamma and that the pharmacology of insulin-sensitizer binding in rodent and human adipocytes is very similar and, moreover, predictive of antihyperglycemic activity in vivo.     

CD4+ lymphocytes provide MUC1-specific tumor immunity in vivo that is undetectable in vitro and is absent in MUC1 transgenic mice

A C57BL/6 mouse transgenic for human MUC1 (MUC1.Tg) was developed to evaluate MUC1-specific tumor immunity in an animal that expresses MUC1 as a normal self protein. Previous studies showed that MUC1.Tg mice, challenged with syngeneic tumors expressing MUC1 (B16.MUC1), developed progressively growing MUC1-positive tumors, whereas wild-type C57BL/6 (wt) mice developed MUC1-negative tumors at a significantly slower rate. The results of a limiting dilution CTL frequency assay were not informative, in that similar numbers of MUC1-specific CTL precursors (CTL) were detected in MUC1.Tg and wt mice. Tumor immunity in vivo was characterized by an adoptive transfer method to evaluate the degree of MUC1 or non-MUC1 tumor immunity in wt or MUC1.Tg mice. The results revealed that wt mice developed protective tumor immunity mediated by MUC1-specific CD4+ lymphocytes, while MUC1.Tg mice were functionally tolerant to MUC1 in vivo. The potential of adoptive immunotherapy to provide immunity to tumors expressing MUC1 and to produce undesirable autoimmunity in recipient MUC1.Tg mice expressing MUC1 as a self Ag was evaluated. Adoptive transfer of immune cells from wt mice primed in vivo with B16.MUC1 tumor cells into MUC1.Tg recipients resulted in significant increases in the survival of MUC1.Tg recipients compared with unmanipulated control MUC .Tg mice challenged with B16.MUC1 tumor cells. This response was specific for MUC1 since control tumors developed at equivalent rates in recipient or control MUC1.Tg mice. No gross or histologic evidence of autoimmunity was observed in recipient MUC1.Tg mice, indicating that tumor immune responses mediated by MUC1-specific CD4+ lymphocytes spare nontransformed epithelia-expressing MUC1.     

Upper airway muscle activity in normal women: influence of hormonal status

Obstructive sleep apnea is a disorder with a strong male predominance. One possible explanation could be an effect of female hormones on pharyngeal dilator muscle activity. Therefore, we determined the level of awake genioglossus electromyogram (EMGgg) and upper airway resistance in 12 pre- and 12 postmenopausal women under basal conditions and during the application of an inspiratory resistive load (25 cmH2O . l-1 . s). In addition, a subgroup of eight postmenopausal women were studied a second time after 2 wk of combined estrogen and progesterone replacement in standard doses. Peak phasic and tonic genioglossus activity, expressed as a percentage of maximum, were highest in the luteal phase of the menstrual cycle (phasic 23.9 +/- 3.8%, tonic 10.2 +/- 1.0%), followed by the follicular phase (phasic 15.5 +/- 2.2%, tonic 7.3 +/- 0.8%), and were lowest in the postmenopausal group (phasic 11.3 +/- 1.6%, tonic of 5.0 +/- 0.6), whereas upper airway resistance did not differ. There was a weak but significant positive correlation between progesterone levels and both peak phasic (P < 0.05) and tonic (P < 0.01) EMGgg. Finally, there was a significant increase in EMGgg in the postmenopausal group restudied after hormone therapy. In conclusion, female hormones (possibly progesterone) have a substantial impact on upper airway dilator muscle activity.     

In vitro culture retards spontaneous activation of cell cycle progression and cortical granule exocytosis that normally occur in in vivo unfertilized mouse eggs

A microchip method has been developed for massive and parallel thermodynamic analyses of DNA duplexes. Fluorescently labeled oligonucleotides were hybridized with oligonucleotides immobilized in the 100 x 100 x 20 mum gel pads of the microchips. The equilibrium melting curves for all microchip duplexes were measured in real time in parallel for all microchip duplexes. Thermodynamic data for perfect and mismatched duplexes that were obtained using the microchip method directly correlated with data obtained in solution. Fluorescent labels or longer linkers between the gel and the oligonucleotides appeared to have no significant effect on duplex stability. Extending the immobilized oligonucleotides with a four-base mixture from the 3'-end or one or two universal bases (5-nitroindole) from the 3'- and/or 5'-end increased the stabilities of their duplexes. These extensions were applied to increase the stabilities of the duplexes formed with short oligonucleotides in microchips, to significantly lessen the differences in melting curves of the AT- and GC-rich duplexes, and to improve discrimination of perfect duplexes from those containing poorly recognized terminal mismatches. This study explored a way to increase the efficiency of sequencing by hybridization on oligonucleotide microchips.     

Stereoselective epoxidation of arachidonic acid by cytochrome P-450s 2CAA and 2C2

In the present study, we determined the stereoselective epoxidation of arachidonic acid by cytochrome P-450 (P-450) 2CAA and P-450 2C2, two arachidonic acid epoxygenases found in rabbit renal cortex, by chiral normal-phase high-performance liquid chromatography (HPLC)-analysis. Purified P-450 2CAA reconstituted with P-450 oxidoreductase, lipid and cytochrome b5 or microsomes isolated from COS-1 cells expressing P-450 2C2 were incubated in the presence of [1-14C]arachidonic acid. The epoxide metabolites 14,15- and 11,12-epoxyeicosatrienoic acids (EETs) were purified by reverse-phase HPLC and derivatized to methyl (14,15-EET) and pentafluorobenzyl (11,12-EET) esters. Enantiomers of 14,15-EET-methyl ester and 11,12-EET-pentafluorobenzyl ester were resolved on Chiralcel OB and OD columns, respectively, with a mobile phase of 0.15% 2-propanol in n-hexane. P-450 2CAA and P-450 2C2 produce 11,12- and 14,15-EETs in distinct ratios but are equally stereoselective at the 11,12-position. P-450 2CAA produced 11(S), 12(R)-EET with 63% stereoselectivity, and P-450 2C2 produced the same enantiomer with 61% stereoselectivity. Both enzymes are also stereoselective at the 14,15- position, preferentially producing the 14(R), 15(S)-EET. P-450 2CAA produces this enantiomer with 72% selectivity, and P-450 2C2 produces it with 62% selectivity. The results of this study indicate that P-450 2CAA and P-450 2C2 are not only regioselective but also exhibit a high degree of stereoselectivity.     

Demonstration of sliding-filter-controlled soliton transmission at20 Gbit/s over 14 Mm

Soliton transmission of a 20 Gbit/s time/polarisation multiplexed signal over 14 Mm path in a recirculating loop with 46.3 km amplification span has been demonstrated using the sliding-filter soliton control technique. Demultiplexing was achieved by using a polarisation-insensitive electroabsorption modulator     

The impact of true partnership between a university Level I trauma center and a community Level II trauma center on patient transfer practices

OBJECTIVE: To examine the effect of a clinical and administrative partnership with an academic urban Level I trauma center on the patient transfer practices at a suburban/rural Level II center. METHODS: Data for 2 years before affiliation (PRE) abstracted from inpatient charts and the trauma registry were compared with that for 2 years after (POST). The following data were collected: number of, reason for, and destination and demographics of transfers. Chi(2) test and t test analyses were used; p < 0.05 defined significance; data are mean +/- SEM. RESULTS: Transfer rate increased from 4% PRE to 6.9% (p = 0.001) POST with no significant difference in age, Glasgow Coma Scale score, Injury Severity Score, or Revised Trauma Score. Repatriation occurred in 12.8% POST (none PRE). The current Level I facility accepted 1.8% of all transfers PRE and 36.4% POST (p = 0.0001). PRE/POST rates by reason are as follows: pediatric, 14.6%/9.0% (p = 0.04); intensive care unit, 0.4%/1.7% (p = 0.13); complex orthopedic, 100%/0% (p = 0.005); vascular, 50%/0% (p = 0.008); spinal cord injury, 100%/100%; and ophthalmologic, 0%/100% (p = 0.005). CONCLUSIONS: In this experience of Level I/II partnership (1) transfer patterns were altered, (2) select patient cohort transfers decreased (pediatric, complex orthopedic, vascular), whereas others increased (aortic work-up), and (3) repatriation rates were low.     

Metastatic thyroid carcinoma arising from congenital goiter due to mutation in the thyroperoxidase gene

A very large cervical tumor that extended to the upper mediastinum was seen in a newborn after an uneventful pregnancy. The computed axial tomography scan confirmed the presence of a solid mass with precise limits and scattered foci of calcifications situated in the anterolateral region of the neck. The infant underwent thyroidectomy on the seventh day after birth. Pathological examination revealed a follicular carcinoma of the thyroid and probable dyshormonogenetic hyperplastic goiter. At 5 months of age, whole body scans indicated the presence of lung and bone metastases, which were treated with therapeutic doses of radioiodine. Genomic DNA was obtained from the newborn, her parents, her paternal aunt, and her paternal grandparents. Denaturing gradient gel electrophoresis analysis of PCR fragments corresponding to exon 14 of the thyroid peroxidase (TPO) gene indicated the presence of a mutant TPO allele present in the propositus, her father, and her paternal grandmother. Sequencing of the TPO gene demonstrated a mutation resulting from an insertion of a single extra cytosine in a stretch of seven cytosines at positions 2505-2511. The insertion caused a frame shift and a stop signal in exon 16. This sequence would translate into a structurally modified and probably inactive TPO protein. We conclude that the aggressive thyroid metastatic carcinoma arose from a dyshormonogenetic goiter caused by a defective TPO protein.