Phosphoproteomic identification of a PDX-1/14-3-3ε interaction in pancreatic beta cells.

Glucose-dependent activation of the homeodomain transcription factor PDX-1 leads to its phosphorylation, to an increase in DNA binding capacity, and to NLS dependent translocation into the nucleus. To uncover unknown mediators of PDX-1 activation, PDX-1 interacting proteins were analysed by pull-down from (32)P-labelled, glucose-stimulated MIN6 cells. Recovered proteins were analysed by 2D gel electrophoresis and mass spectrometry. We identified 14-3-3ε as a novel PDX-1 binding protein and confirmed the interaction in vivo by Fluorescence Resonance Energy Transfer (FRET) analysis. We propose that 14-3-3ε interacts directly with PDX-1 to regulate its cellular distribution in pancreatic beta cells.

[Clinical accuracy of fluoroscopic navigation at the thoracic and lumbar spine]. / Klinische Präzision der C-Arm-Navigation an der Brust- und Lendenwirbelsäule.

The aim of the present study was the verification of the accuracy of 2D fluoroscopy-based navigated pedicle screw placements at the thoracic and lumbar spine in a case series of traumatised patients. Within 36 months 111 pedicle screws were instrumented using C-arm based navigation in 29 patients, 60 at the thoracic and 51 at the lumbar spine. All screw positions were evaluated postoperatively by a routine thin-slice CT scan using multiplanar reconstruction. The position of a screw in relation of its pedicle was classified as: a) screw completely intraosseous, b) screw perforated less than thread level and c) screw perforated over thread level. In 34 thoracic (56.7%) and 32 lumbar (62.7%) screws complete intraosseous placement was observed, 14 thoracic screws (23.3%) and 14 lumbar screws (27.5%) perforated less than thread level. Perforations over thread level were found in 12 thoracic (20%) and 5 lumbar (9.8%) screws. Only medial and lateral perforations of the pedicle were documented (without neurological signs), cranial or caudal perforations did not occur. Segmentation of the C-arm navigation into two comparable treatment periods showed a learning curve with a reduction of perforations in the second sequence (after 57 pedicle instrumentations) of about 15%, this was not found to be statistically significant. The fluoroscopic navigation of pedicle screws is a safe procedure at the lumbar spine with equal accuracy compared to the non-navigated conventional instrumentation. Application of C-arm navigation at the thoracic spine showed more inaccuracies, so that 3D-based navigation seems to be more advantageous in this region.

[Clinical accuracy of fluoroscopic navigation at the thoracic and lumbar spine]. / Klinische Präzision der C-Arm-Navigation an der Brust- und Lendenwirbelsäule.

The aim of the present study was the verification of the accuracy of 2-D fluoroscopy-based navigated pedicle screws at the thoracic and lumbar spine in a case series of traumatised patients. Within 36 months 111 pedicle screws in 29 patients were instrumented using C-arm based navigation, 60 at the thoracic and 51 at the lumbar spine. All screw positions were evaluated postoperatively by a routine thin slice CT scan using multiplanar reconstruction. The position of a screw in relation to its pedicle was classified in a) screw completely intraosseous, b) screw perforated less than thread level and c) screw perforated more than thread level. In 34 thoracic (56.7%) and 32 lumbar (62.7%) screws complete intraosseous placement was observed, 14 thoracic (23.3%) and 14 lumbar (27.5%) screws perforated less than thread level. Perforations more than thread level were found in 12 thoracic (20%) and 5 lumbar (9.8%) screws. Only medial and lateral perforations of the pedicle were documented (without neurological signs), cranial or caudal ones did not occur. Segmentation of the C-arm navigation into two comparable treatment periods showed a learning curve with a reduction of perforations in the second sequence (after 57 pedicle instrumentations) of about 15%, this was found to be not statistically significant. The fluoroscopic navigation of pedicle screws is a safe procedure at the lumbar spine with equal accuracy compared to the non-navigated conventional instrumentation. Application of C-arm navigation at the thoracic spine showed more inaccuracies, so that 3-D-based navigation seems to be advantageous in this region.

[Establishing a protein signature from prostate tissue biopsies]. / Erstellung eines Proteinprofils aus Gewebeproben der Prostata.

The prostate-specific antigen test (PSA) has been a major factor contributing to a better management of prostate cancer. The low specificity limits its use in diagnosis especially in early detection of prostate cancer. Multiply expressed proteins need to be identified to establish a disease-specific protein signature that distinguishes between cancerous and noncancerous tissue. The first aim of our study is to identify differentially expressed proteins in both tissues using two-dimensional gel electrophoresis and subsequent mass spectrometry. We elucidated whether prostate biopsies are useful. First results have shown a different protein expression pattern in cancerous and noncancerous tissue. PCR revealed an increasing amount of mRNA for some upregulated proteins. We conclude that biopsies are useful material to establish protein expression patterns.

Expression of annexin AI in conventional renal cell carcinoma (CRCC) correlates with tumour stage, Fuhrman grade, amount of eosinophilic cells and clinical outcome.

There is increasing evidence that Annexin AI (ANX AI) expression is dysregulated in several carcinomas and tumour cell lines. In order to gain insight into the putative role of ANX AI in tumorigenesis, clinical outcome and metastatic potential of conventional renal cell carcinomas (CRCCs) we investigated the expression of ANX AI in CRCCs and metastases. Furthermore, it was elucidated whether ANX AI overexpression affects migratory potential in Caki-1 cells. ANX AI immunohistochemistry was performed on 33 samples of CRCCs and 10 metastases. ANX AI expression was assessed in 12 samples by 2-dimensional gelelectrophoresis (2-DE), subsequent mass spectrometry and RT-PCR. Immunohistochemical data were statistically correlated with pathological parameters, amount of eosinophilic cells and clinical outcome. Furthermore, a haptotactic migration assay was done on Caki-1 cells transfected with ANX AI. Immunostaining for ANX AI was found in 18 tumours and all metastases investigated. Intensity of immunohistochemical staining correlated to Fuhrman grade, amount of eosinophilic cells and clinical outcome. 2-DE and RT-PCR confirmed the presence of ANX AI in neoplastic tissue. Overexpression of ANX AI did not significantly influence cell migration. From these findings ANX AI expression seems to be related to Fuhrman grade, clinical outcome and metastatic potential of CRCCs. Thus ANX AI could serve as a prognostic marker for tumour progression.

[Precision of navigation-assisted surgery of the thoracic and lumbar spine]. / Präzision der navigationsgestützten Chirurgie an der Brust- und Lendenwirbelsäule.

The goal of these studies was to evaluate the accuracy of in vivo and in vitro application of CT- and C-arm-based navigation at the thoracic and lumbar spine. With CT based navigation, 82 pedicle screws were consecutively inserted, 53 into the thoracic and 29 into the lumbar spine. Seven (13%) perforations were detected at the thoracic spine and two (7%) at the lumbar spine. Additionally, minor perforations below the thread depth were seen in six (11%) thoracic and in two (7%) lumbar instrumentation. With C-arm-based navigation, 74 screws were consecutively placed into 38 thoracic and 36 lumbar pedicles. Perforations were noted in ten (26%) thoracic and four (11%) lumbar implants. Minor perforations were observed in another nine (24%) thoracic and ten (28%) lumbar pedicles. The observer-independent and standardized in vitro study based on a transpedicular 3.2-mm drill hole aiming a 4-mm steel ball in a plastic bone model showed pedicle perforations of the drill canal only in thoracic vertebrae, 1 of 15 in CT-based and 3 of 15 in C-arm navigation. The quantitative calculation of the smallest distance between the central line through the drill canal and the center of the steel ball resulted in 1.4 mm (0.5-4.8 mm) for the CT-based navigation at the thoracic spine and in 1.8 mm (0.5-3 mm) at the lumbar spine. For the C-arm based navigation the distance was 2.6 mm (0.9-4.8 mm) for the thoracic spine and 2 mm (1.2-3 mm) for the lumbar spine. In our opinion, the clinical results of the comparative accuracy of CT- and C-arm-based navigation in the present study showed moderate advantages of the CT-based technique in the thoracic spine, whereas CT- and C-arm based navigation had comparable perforation rates at the lumbar pedicle. The results of the experimental study correlated with the clinical data.

Bis(1H-2-indolyl)-1-methanones as inhibitors of the hematopoietic tyrosine kinase Flt3.

Aberrant expression and activating mutations of the class III receptor tyrosine kinase Flt3 (Flk-2, STK-1) have been linked to poor prognosis in acute myeloid leukemia (AML). Inhibitors of Flt3 tyrosine kinase activity are, therefore, of interest as potential therapeutic compounds. We previously described bis(1H-2-indolyl)-1-methanones as a novel class of selective inhibitors for platelet-derived growth factor receptors (PDGFR). Several bis(1H-2-indolyl)-1-methanone derivatives, represented by the compounds D-64406 and D-65476, are also potent inhibitors of Flt3. They inhibit proliferation of TEL-Flt3-transfected BA/F3 cells with IC(50) values of 0.2-0.3 microM in the absence of IL-3 but >10 microM in the presence of IL-3. Ligand-stimulated autophosphorylation of Flt3 in EOL-1 cells and corresponding downstream activation of Akt/PKB are effectively inhibited by bis(1H-2-indolyl)-1-methanones whereas autophosphorylation of c-Kit/SCF receptor or c-Fms/CSF-1 receptor is less sensitive or insensitive, respectively. Flt3 kinase purified by different methods is potently inhibited in vitro, demonstrating a direct mechanism of inhibition. 32D cells, expressing a constitutively active Flt3 variant with internal tandem duplication are greatly sensitized to radiation-induced apoptosis in the presence of D-64406 or D-65476 in the absence but not in the presence of IL-3. Thus, bis(1H-2-indolyl)-1-methanones are potential candidates for the treatment of Flt3-driven leukemias.

Pyrrolo[3,4-c]-beta-carboline-diones as a novel class of inhibitors of the platelet-derived growth factor receptor kinase.

Members of the structurally diverse family of beta-carbolines have previously been shown to exhibit a wide range of biological activities. A novel synthetic strategy for generation of beta-carbolines was developed, allowing imido-beta-carbolines to be created in three steps from known compounds. The compounds were screened for inhibition of platelet-derived growth factor (PDGF)-stimulated tyrosine phosphorylation in Swiss 3T3 fibroblasts. A number of the newly synthesized beta-carbolines with moderate to potent inhibitory activity were revealed. The most active derivative, 2,3-dihydro-8,9-dimethoxy-5-(2-methylphenyl)-1H,6H-pyrrolo[3, 4-c]pyrido¿3,4-bindole-1,3-dione 2ee, inhibited purified PDGF receptor kinase and PDGF-receptor autophosphorylation in intact cells with IC(50) values of 0.4 and 2.6 microM, respectively. Dione 2ee also inhibited PDGF-stimulated DNA synthesis in Swiss 3T3 fibroblasts with an IC(50) of 3.2 microM. The compound had no effect on Src or epidermal growth factor (EGF) receptor kinase activity and a six-seven-fold higher IC(50) for inhibition of basic fibroblast growth factor (bFGF)-stimulated tyrosine phosphorylation or Kit/stem cell factor (SCF) receptor autophosphorylation, indicating a reasonable extent of kinase specificity. Thus, beta-carbolines present a new lead of tyrosine kinase inhibitors with the capacity to selectively interfere with PDGF receptor signal transduction and PDGF-dependent cell growth.

Purification and characterization of a fully active recombinant tobacco cytosolic NADP-dependent isocitrate dehydrogenase in Escherichia coli: evidence for a role for the N-terminal region in enzyme activity.

The recently isolated full-length NADP-dependent isocitrate dehydrogenase (ICDH) cDNA encoding the tobacco cytosolic isoenzyme has been cloned into the expression vector pET8c and used to transform Escherichia coli strain BL21 (DE3). The recombinant protein was purified to electrophoretic homogeneity and used to raise polyclonal antibodies. Its kinetic properties were found to be identical to those of the cytosolic ICDH isoenzyme purified from tobacco cell cultures. The recombinant and the endogenous bacterial ICDH could be easily distinguished by their different behaviors during anion-exchange column chromatography and immunological response. An incomplete ICDH-encoding cDNA clone, encoding a protein lacking the first 36 amino acids at the N-terminus, was cloned into the expression vector pKK233-2 and used to transform ICDH-lacking E. coli cells (strain 2004). The truncated, recombinant ICDH produced by the bacteria was found to be inactive.

Influence of human serum albumin content in formulations on the bioequivalency of interferon alfa-2a given by subcutaneous injection in healthy male volunteers.

To determine the influence of human serum albumin (HSA) content in formulations on the bioequivalency of recombinant interferon alfa-2a, a double-blind, randomized, two-way crossover study was conducted in 24 healthy male volunteers. Subjects received a single subcutaneous injection of 18 million IU of Roferon-A reconstituted with either the diluent containing 10 mg of HSA or the HSA-free diluent; final HSA contents in the 2 formulations were 15 mg and 5 mg, respectively. Administration of the 2 formulations resulted in similar 48-hour Roferon-A serum concentration-time profiles and comparable frequency and intensity of adverse events. The statistical analysis using the two one-sided tests procedure showed that both formulations were bioequivalent for pharmacokinetic parameters such as Cmax, tmax, AUC48, and AUC. We conclude that a threefold change in HSA content in formulations does not alter the bioequivalency of Roferon-A.

The pharmacokinetic-pharmacodynamic (Digit Symbol Substitution Test) relationship of flumazenil in a midazolam steady-state model in healthy volunteers.

To characterize the plasma concentration-effect relationship of flumazenil in the presence of a predefined midazolam level, a double-blind, placebo-controlled, randomized two-way crossover study was conducted in nine healthy male subjects. After reaching a criterion level of midazolam-induced depression of the Digit Symbol Substitution Test (DSST), volunteers received a dose of flumazenil (1.0 mg) or placebo over 1 minute, with the infusion of midazolam continued. Blood samples were collected, simultaneously with the DSST assessment, at predetermined intervals and were assayed for flumazenil and/or midazolam plasma concentrations. Pharmacokinetic-pharmacodynamic modeling techniques were used to estimate the equilibration rate constant (keo) between plasma concentration and effect for flumazenil; a sigmoidal maximum-effect model was used to relate the DSST score to the flumazenil plasma concentration. Flumazenil exhibited a rapid onset (the half-life of equilibration between drug concentration in the blood and drug effect was 3.3 minutes) and short duration of action (the flumazenil plasma concentration causing half-maximal effect was 7.4 ng/ml, which was reached about 1 hour after dosing). The results of this study also show the competitive nature of flumazenil as a midazolam antagonist.

Dose-dependent and time-dependent pharmacokinetics in the dog after intravenous administration of dextrorphan.

The disposition of dextrorphan after single ascending iv doses and multiple iv dosing regimens was studied in Marshall beagle dogs. A dose-dependent decrease in plasma clearance was observed after the administration of single iv doses of 0.88 mg/kg, 2.64 mg/kg, and 8.8 mg/kg of dextrorphan (i.e. mean plasma clearance values +/- SD were 100 +/- 25 vs. 68 +/- 28 vs. 48 +/- 20 ml/min.kg, respectively; p less than 0.001). Upon multiple dosing, the plasma clearance of dextrorphan increased in a time-dependent fashion for the two highest doses, approaching values observed for the 0.88 mg/kg/day iv dosing regimen. Female dogs exhibited a greater increase in plasma clearance with time. For all dogs, however, dextrorphan plasma clearance approached or exceeded hepatic plasma flow rate, suggesting the possibility of extrahepatic metabolism or elimination. Modest dose- and time-dependent changes in the steady-state volume of distribution of dextrorphan also were observed. The AUC of the conjugated metabolites of dextrorphan decreased in a time-dependent manner for the 8.8 mg/kg/day dosing regimen. The nonlinear kinetics of dextrorphan after iv administration appeared to occur only after potentially toxic dosing regimens of dextrorphan hydrochloride. We postulate mechanisms to explain the dose- and time-dependent kinetics of dextrorphan observed in the beagle dog.

Synthesis and peripheral cardiovascular action of cis- and trans-2-(3,4-dimethoxybenzyl)cyclopentylamine hydrochlorides.

The hydrochlorides of cis- and trans-2-(3,4-dimethoxybenzyl)cyclopentylamine have been synthesized. They are transient hypotensive agents with early and delayed depressor effects and antagonists of dopamine-induced vasodepression. In the atropinized and phenoxybenzamine-treated dog, the threshold dose for hypotension was 3-5 mumol/kg. The early depressor phases were attenuated variably by different types of antagonists, suggesting a nonspecific interaction with blood pressure regulation mechanisms. Cimetidine blocked the delayed depressor phases, consistent with endogenous histamine release. The cis amine hydrochloride was three to four times more potent than its trans isomer as a peripheral dopamine blocking agent. Cimetidine but not diphenhydramine interfered with this effect.