Noninvasive imaging of cell proliferation following mitogenic extracellular kinase inhibition by PD0325901

The mitogenic extracellular kinase 1/2 (MEK1/2) inhibitor, PD0325901, has potent activity in a number of cancer cell types in vitro. In SKMEL-28 human melanoma cells (BRAF mutant), the drug rapidly decreased phosphorylated extracellular signal-regulated kinase 1/2, cyclin D1, and thymidine kinase 1 protein levels. We investigated if 3'-deoxy-3'-[(18)F]fluorothymidine-positron emission tomography ([(18)F]FLT-PET) could be used to image changes in cell proliferation following MEK1/2 inhibition in vivo. Mice bearing SKMEL-28 and human colon cancer HCT116 (K-RAS mutant) xenografts were treated daily with PD0325901 at 25 mg/kg and imaged by dynamic [(18)F]FLT-PET after 1 and 10 days of initiating treatment. The drug decreased tumor [(18)F]FLT uptake after 1 and 10 days of treatment compared with control animals. The normalized (maximal) [(18)F]FLT uptake in SKMEL-28 xenografts (at 60 minutes; NUV(max)) after 1 day of vehicle or PD0325901 therapy was 1.81 +/- 0.18 versus 1.23 +/- 0.10, respectively (P = 0.03). In this model, NUV(max) after 10 days was 2.07 +/- 0.40 versus 1.08 +/- 0.14, respectively (P = 0.03). The corresponding values for HCT116 tumors were 2.30 +/- 0.84 versus 1.88 +/- 0.36 (P = 0.045) after 1 day, and 1.97 +/- 0.13 versus 1.00 +/- 0.03 (P = 0.03) after 10 days. Similar changes were found for other [(18)F]FLT retention variables. The drug decreased phosphorylated extracellular signal-regulated kinase 1/2, cyclin D1, and thymidine kinase 1 protein. Tumor [(18)F]FLT-PET variables correlated with proliferation as measured by Ki67 labeling index (r >/= 0.6; P >/= 0.003). In summary, [(18)F]FLT-PET is a sensitive imaging biomarker for detecting the antiproliferative effect of MEK1/2 inhibition by PD0325901. [Mol Cancer Ther 2008;7(9):3112-21].     

Three-photon microscopy shows that somatic release can be a quantitatively significant component of serotonergic neurotransmission in the mammalian brain

Recent experiments on monoaminergic neurons have shown that neurotransmission can originate from somatic release. However, little is known about the quantity of monoamine available to be released through this extrasynaptic pathway or about the intracellular dynamics that mediate such release. Using three-photon microscopy, we directly imaged serotonin autofluorescence and investigated the total serotonin content, release competence, and release kinetics of somatic serotonergic vesicles in the dorsal raphe neurons of the rat. We found that the somata of primary cultured neurons contain a large number of serotonin-filled vesicles arranged in a perinuclear fashion. A similar distribution is also observed in fresh tissue slice preparations obtained from the rat dorsal raphe. We estimate that the soma of a cultured neuron on an average contains about 9 fmoles of serotonin in about 450 vesicles (or vesicle clusters) of < or =370 nm average diameter. A substantial fraction (>30%) of this serotonin is released with a time scale of several minutes by K(+)-induced depolarization or by para-chloroamphetamine treatment. The amount of releasable serotonin stored in the somatic vesicles is comparable to the total serotonin content of all the synaptic vesicles in a raphe neuron, indicating that somatic release can potentially play a major role in serotonergic neurotransmission in the mammalian brain.     

Effect of Bauhinia racemosa stem bark on N-nitrosodiethylamine-induced hepatocarcinogenesis in rats

The aim of this study is to investigate the antioxidant defense system induced by the methanol extract of Bauhinia racemosa L.(MEBR) against N-nitrosodiethylamine (NDEA)-induced hepatocarcinogenesis in Wister albino rats. The effects of MEBR on surface visible macroscopic (Morphometry) liver lesions (neoplastic nodules) and the levels of serum enzymes, lipid peroxidation and antioxidants were evaluated in NDEA-induced hepatocarcinogenesis in rats. In rats treated, with NDEA, significantly elevated levels of serum enzymes (SGOT, SGPT and ALP), bilirubin and decreased levels of protein and uric acid were observed. Significantly elevated amount of malondialdehyde (MDA), the end product of lipidperoxidation, indicated higher levels of lipid peroxidation, which was accompanied by significantly decreased levels of antioxidants like vitamin C, vitamin E, reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT). Administration of MEBR was able to suppress nodule development/hepatocellular lesion formation in rats. The extract treatment increases in antioxidant levels and dramatic decreases in lipid peroxidation levels. MEBR also produced a protective effect by decreasing the level of serum enzymes, bilirubin and increased the protein and uric acid levels. The results suggest that MEBR exert chemopreventive effects by suppressing nodule development and decreasing lipid peroxidation and enhancing the levels of antioxidants in NDEA carcinogenesis by reducing the formation of free radicals.     

Antimycobacterial activities of novel 1-(cyclopropyl/tert-butyl/4-fluorophenyl)-1,4-dihydro- 6-nitro-4-oxo-7-(substituted secondary amino)-1,8-naphthyridine-3-carboxylic acid

Fifty-one 1-(cyclopropyl/tert-butyl/4-fluorophenyl)-1,4-dihydro-6-nitro-4-oxo-7-(substituted secondary amino)-1,8-naphthyridine-3-carboxylic acids were synthesized and evaluated for antimycobacterial in vitro and in vivo against Mycobacterium tuberculosis H37Rv (MTB), multi-drug-resistant Mycobacterium tuberculosis (MDR-TB) and Mycobacterium smegmatis (MC2) and also tested for the ability to inhibit the supercoiling activity of DNA gyrase from M. smegmatis. Among the synthesized compounds, 1-tert-butyl-1,4-dihydro-7-(4,4-dimethyloxazolidin-3-yl)-6-nitro-4-oxo-1,8-naphthyridine-3-carboxylic acid (10q) was found to be the most active compound in vitro with an MIC of 0.1 microM against MTB and MDR-TB and was 3 and 455 times more potent than isoniazid against MTB and MDR-TB, respectively. In the in vivo animal model 10q decreased the bacterial load in lung and spleen tissues with 2.39 and 3.89-log10protections respectively at the dose of 50 mg/kg body weight.     

Oocyte and follicular fluid characteristics in women with mild endometriosis

The follicular development, fertilization and cleavage rates and follicular fluid endocrinology were assessed in 20 women with mild endometriosis during a spontaneous menstrual cycle. Diagnostic laparoscopy was performed 32 h after the onset of the endogenous luteinizing hormone (LH) surge in the 20 women with laparoscopically confirmed mild endometriosis and in a control group of ten women with tubal infertility. There was no significant difference in oocyte maturity or fertilization and cleavage rates between the women with mild endometriosis and those in the control group. The endocrine milieu of the pre-ovulatory oocyte at the time of aspiration was similar in the two groups.     

Platelet-leukocyte-endothelial cell interactions after middle cerebral artery occlusion and reperfusion.

The adhesion of both leukocytes and platelets to microvascular endothelial cells has been implicated in the pathogenesis of ischemia/reperfusion (I/R) injury in several vascular beds. The objectives of this study were to (1) assess the platelet-leukocyte-endothelial cell interactions induced in the cerebral microvasculature by middle cerebral artery occlusion (MCAO)/reperfusion, and (2) define the molecular determinants of the prothrombogenic and inflammatory responses in this model of focal I/R. MCAO was induced for 1 hour in wild-type (WT) mice, WT mice treated with a monoclonal antibody (mAb) to either P-selectin or GPIIb/IIIa, and in P-selectin-/-(P-sel-/-) chimeras. Isolated platelets labeled with carboxyfluorescein diacetate succinimidyl ester (CFDASE) were administered intravenously and observed with intravital fluorescence microscopy. Leukocytes were observed after intravenous injection of rhodamine 6G. One hour of MCAO followed by 1 hour of reperfusion resulted in the rolling and adhesion of leukocytes in venules, and after 4 hours of reperfusion, the adhesion of both leukocytes and platelets was detected. Although both the P-selectin and GPIIb/IIIa mAbs significantly reduced the adhesion of leukocytes and platelets at 4 hours of reperfusion, the antiadhesive effects of the P-selectin mAb were much greater. The leukocyte and platelet adhesion responses were significantly attenuated in both P-sel-/- --> WT and WT --> P-sel-/- bone marrow chimeras, compared with WT --> WT chimeras. Neutropenia, induced by antineutrophil serum treatment, also reduced the recruitment of leukocytes and platelets after cerebral I/R. These findings implicate a major role for both platelet-associated and endothelial cell-associated P-selectin, as well as neutrophils in the inflammatory and prothrombogenic responses in the microcirculation after focal cerebral I/R.     

Synthesis and biological evaluation of hydroxamate-based iron chelators

A new and versatile route to desferrioxamine B (DFO, 1) is described. Hydroxamate reagent 4 was elaborated in a series of high yield steps to the tert-butoxycarbonyl nitrile 11, which provided DFO in three transformations. The intermediate 11 could also be utilized in the preparation of DFO analogues which contain terminal N-acyl groups other than acetyl. The methodology was further employed in the syntheses of the DFO polyether analogues 2 and 3, beginning with the 3,6,9-trioxadecylation of N-(tert-butoxycarbonyl)-O-benzylhydroxylamine. Polyethers 2 and 3 are neutral molecules, which are somewhat more lipophilic than the parent DFO. Polyether hydroxamate 2 was shown to be nearly 3 times as effective as desferrioxamine at clearing iron in rats.     

Gartner duct cyst in pregnancy presenting as a prolapsing pelvic mass

Gartner duct cysts are the remnants of the Wolffian duct and they are rarely seen in adulthood. We present a case of a pregnant patient with a prolapsing vaginal mass. A diagnosis of Gartner duct cyst was made after MRI was performed. The Gartner duct cyst was drained when the patient went into labour allowing vaginal delivery to be performed.     

Relationship between stimulation train characteristics and dynamic human skeletal muscle performance

Aim: The purpose of the present study was to investigate the effect of activation frequency on dynamic human muscle performance for a range of train durations and number of pulses during free limb movement. Methods: The quadriceps femoris muscles of 10 subjects were activated with stimulation trains with different activation frequency, train durations and number of pulses. The peak excursion produced in response to each train was the dependent measure of muscle performance. Results: The excursion–frequency (for a 300‐ms train duration) and excursion–train duration (for trains with frequencies of 10, 30 or 59 Hz) relationships could each be fit with a two‐parameter exponential equation (R² values > 0.97). Because the number of pulses in a stimulation train is a function of both train duration and frequency, the excursion produced as a function of the number of pulses was characterized by a three‐parameter exponential equation that represented this combined relationship. The relationship between the measured and predicted excursions in response to a wide range of stimulation trains had a R² = 0.96. In addition, one‐way repeated measures analyses of variance (anova s) showed that the frequency at which the maximum excursion was produced increased with an increase in the number of pulses in the trains tested. Conclusion: These results show the importance of train duration and the number of pulses contained within a train on the relationship between activation frequency and human skeletal muscle performance.