Combining Click Chemistry-Based Proteomics With Dox-Inducible Gene Expression.

Inactivating mutations in single genes can trigger, prevent, promote, or alleviate diseases. Identifying such disease-related genes is a main pillar of medical research. Since proteins play a crucial role in mediating these effects, their impact on the diseased cells' proteome including posttranslational modifications has to be elucidated for a detailed understanding of the role of these genes in the disease process. In complex disorders, like cancer, several genes contribute to the disease process, thereby hampering the assignment of a proteomic change to the corresponding causative gene. To enable comprehensive screening for the impact of inactivation of a gene, e.g., loss of a tumor suppressor in cancer, on the cellular proteome, we present a strategy based on combination of three technologies that is recombinase-mediated cassette exchange, click chemistry, and mass spectrometry. The methodology is exemplified by the analysis of the proteomic changes induced by the loss of a tumor suppressor gene in colorectal cancer cells. To demonstrate the applicability to screen for posttranslational modification changes, we also describe the analysis of protein glycosylation changes caused by the tumor suppressor inactivation. In principle, this strategy can be applied to analyze the effects of any gene of interest on protein expression as well as posttranslational modification by glycosylation. Moreover adaptation of the strategy to an appropriate cell culture model has the potential for application on a broad range of diseases where the disease-promoting mutations have been identified.

Molecular architecture of the DED chains at the DISC: regulation of procaspase-8 activation by short DED proteins c-FLIP and procaspase-8 prodomain.

The CD95/Fas/APO-1 death-inducing signaling complex (DISC), comprising CD95, FADD, procaspase-8, procaspase-10, and c-FLIP, has a key role in apoptosis induction. Recently, it was demonstrated that procaspase-8 activation is driven by death effector domain (DED) chains at the DISC. Here, we analyzed the molecular architecture of the chains and the role of the short DED proteins in regulating procaspase-8 activation in the chain model. We demonstrate that the DED chains are largely composed of procaspase-8 cleavage products and, in particular, of its prodomain. The DED chain also comprises c-FLIP and procaspase-10 that are present in 10 times lower amounts compared with procaspase-8. We show that short c-FLIP isoforms can inhibit CD95-induced cell death upon overexpression, likely by forming inactive heterodimers with procaspase-8. Furthermore, we have addressed mechanisms of the termination of chain elongation using experimental and mathematical modeling approaches. We show that neither c-FLIP nor procaspase-8 prodomain terminates the DED chain, but rather the dissociation/association rates of procaspase-8 define the stability of the chain and thereby its length. In addition, we provide evidence that procaspase-8 prodomain generated at the DISC constitutes a negative feedback loop in procaspase-8 activation. Overall, these findings provide new insights into caspase-8 activation in DED chains and apoptosis initiation.

Calcium-signaling networks in olfactory receptor neurons.

The olfactory neuroepithelium represents a unique interface between the brain and the external environment. Olfactory function comprises a distinct set of molecular tasks: sensory signal transduction, cytoprotection and adult neurogenesis. A multitude of biochemical studies has revealed the central role of Ca(2+) signaling in the function of olfactory receptor neurons (ORNs). We set out to establish Ca(2+)-dependent signaling networks in ORN cilia by proteomic analysis. We subjected a ciliary membrane preparation to Ca(2+)/calmodulin-affinity chromatography using mild detergent conditions in order to maintain functional protein complexes involved in olfactory Ca(2+) signaling. Thus, calmodulin serves as a valuable tool to gain access to novel Ca(2+)-regulated protein complexes. Tandem mass spectrometry (nanoscale liquid-chromatography-electrospray injection) identified 123 distinct proteins. Ninety-seven proteins (79%) could be assigned to specific olfactory functions, including 32 to sensory signal transduction and 40 to cytoprotection. We point out novel perspectives for research on the Ca(2+)-signaling networks in the olfactory system of the rat.

Proteomic analysis reveals successive aberrations in protein expression from healthy mucosa to invasive head and neck cancer.

Development of head and neck squamous cell carcinoma (HNSCC) is a multistep process and in many cases involves a phenomenon coined 'field cancerization'. In order to identify changes in protein expression occurring at different stages of tumorigenesis and field cancerization, we analysed 113 HNSCCs and 73 healthy, 99 tumor-distant and 18 tumor-adjacent squamous mucosae by SELDI-TOF-MS on IMAC30 ProteinChip Arrays. Forty-eight protein peaks were differentially expressed between healthy mucosa and HNSCC. Calgizarrin (S100A11), the Cystein proteinase inhibitor Cystatin A, Acyl-CoA-binding protein, Stratifin (14-3-3 sigma), Histone H4, alpha- and beta-Hemoglobin, a C-terminal fragment of beta-hemoglobin and the alpha-defensins 1-3 were identified by mass spectrometry. The alpha-defensins showed various alterations in expression as validated by immunohistochemistry (IHC). Supervised prediction analysis revealed excellent classification of healthy mucosa (94.5% correctly classified) and tumor samples (92.9% correctly classified). Application of this classifier to the tumor-adjacent and tumor-distant mucosa samples disclosed dramatic changes: only 59.6% of the tumor-distant biopsies were classified as normal, 27.3% were predicted as aberrant or HNSCC. Strikingly, 72% of the tumor-adjacent mucosae were predicted as aberrant. These data provide evidence for the existence of genetically altered fields with inconspicuous histology. Comparison of the protein profiles in the tumor-distant-samples with clinical outcome of 32 patients revealed a significant association between aberrant profiles with tumor relapse events (P=0.018; Fisher's exact test, two-tailed). We conclude that proteomic profiling in conjunction with protein identification greatly outperforms histopathological diagnosis and may have significant predictive power for clinical outcome and personalized risk assessment.

Carbon monoxide production from desflurane, enflurane, halothane, isoflurane, and sevoflurane with dry soda lime.

BACKGROUND: Previous studies in which volatile anesthetics were exposed to small amounts of dry soda lime, generally controlled at or close to ambient temperatures, have demonstrated a large carbon monoxide (CO) production from desflurane and enflurane, less from isoflurane, and none from halothane and sevoflurane. However, there is a report of increased CO hemoglobin in children who had been induced with sevoflurane that had passed through dry soda lime. Because this clinical report appears to be inconsistent with existing laboratory work, the authors investigated CO production from volatile anesthetics more realistically simulating conditions in clinical absorbers. METHODS: Each agent, 2.5 or 5% in 2 l/min oxygen, were passed for 2 h through a Dräger absorber canister (bottom to top) filled with dried soda lime (Drägersorb 800). CO concentrations were continuously measured at the absorber outlet. CO production was calculated. Experiments were performed in ambient air (19-20 degrees C). The absorbent temperature was not controlled. RESULTS: Carbon monoxide production peaked initially and was highest with desflurane (507 +/- 70, 656 +/- 59 ml CO), followed by enflurane (460 +/- 41, 475 +/- 99 ml CO), isoflurane (176 +/- 2.8, 227 +/- 21 ml CO), sevoflurane (34 +/- 1, 104 +/- 4 ml CO), and halothane (22 +/- 3, 20 +/- 1 ml CO) (mean +/- SD at 2.5 and 5%, respectively). CONCLUSIONS: The absorbent temperature increased with all anesthetics but was highest for sevoflurane. The reported magnitude of CO formation from desflurane, enflurane, and isoflurane was confirmed. In contrast, a smaller but significant CO formation from sevoflurane was found, which may account for the CO hemoglobin concentrations reported in infants. With all agents, CO formation appears to be self-limited.

[Acetyl starch as volume substitute, a possible alternative to HES]. / Acetylstärke als Volumenersatz, eine mögliche Alternative zu HES.

OBJECTIVES: Pharmacokinetics and tolerability of acetyl starch (ACS) in comparison to hydroxyethyl starch (HES) were investigated after repeated intravenous infusions. METHODS AND COHORTS: A 500 ml solution of ACS (n = 8) or HES (n = 9) was infused to male volunteers (Age 25-42 Years) over four hours on five consecutive days. RESULTS: Comparing the pharmacokinetic parameters, marked differences were found between ACS and HES. A continuos increase of Cmax, AUC0-24 and t1/2 over the five days caused by administration of HES was due to an accumulation of HES in serum. However, after administration of ACS all these parameters remained unaltered. The repeated infusion of 50 g ACS did not cause any changes of the acid-base-status. The influence of ACS on the coagulation parameters was comparable to that of HES and due to dilution effects. The acetic acid concentration increased up to 2.96 +/- 0.67 mg/dl following ACS infusion. The blood glucose concentration was not influenced by the infusion of HES or of ACS. The repeated ACS infusions were well tolerated. In contrast to HES, ACS did not accumulate in serum. CONCLUSION: According to these data ACS is an alternative to HES for volume replacement. Well-known side effects due to long storage of HES in tissues may not occur following application of ACS. However, the wide usage of ACS is restricted by the limited stability of ACS solutions at room temperature. ACS solutions are thus only stable during storage at lower temperatures.

[The use of lithium hydroxide for carbon dioxide absorption prevents formation of compound A during sevoflurane anesthesia]. / Die Anwendung von Lithiumhydroxid als Kohlendioxidabsorbens verhindert das Entstehen von Compound A während Sevoflurananästhesie.

UNLABELLED: Aim of the study was the clinical investigation of sevoflurane degradation when using water-free lithiumhydroxide versus moist Drägersorb 800 for carbon dioxide absorption. METHODS: Concentrations of Compound A in the inspiratory gas mix and serum fluoride levels were measured in two groups of 8 patients each. RESULTS: When water-free lithiumhydroxide was used for carbon dioxide absorption, concentration of Compound A in the inspiratory gas mix was ca. 1 ppm (near minimal level of detection) as compared to ca. 20 ppm for moist Drägersorb 800. The concentration of fluoride increased during sevoflurane anesthesia (15.0 +/- 4.8 mumol/l with lithiumhydroxide versus 21.9 +/- 4.0 mumol/l with Drägersorb 800 after 60 mins). CONCLUSIONS: When lithiumhydroxide is used, there is only minimal formation of compound A from sevoflurane degradation. Since serum fluoride levels increased in both patient groups, we conclude that this is caused mainly by metabolism of sevoflurane. Capacity of lithiumhydroxide for carbon dioxide absorption is similar to that of Drägersorb 800. Therefore, the use of lithiumhydroxide increases patient safety.

[Various reactions of sevoflurane with the individual components of soda lime]. / Unterschiedliche Reaktion von Sevofluran mit einzelnen Komponenten von Atemkalk.

The various components of commercial soda lime (sodium hydroxide, potassium hydroxide, calcium hydroxide, barium hydroxide) were studied in terms of their reactivity with sevoflurane at its boiling point (59 degrees C). A simple closed system, a reflux cooler, served as a model. Analyses were performed by GC/MS. Besides sevoflurane, we identified four compounds: A, B, C, and D. Free methanol, formaldehyde and formic acid could not be found. Presumably methanol is transferred from an intermediate formalin-semiacetal of the hexafluorisopropanol. Calcium hydroxide and barium hydroxide showed little reaction with sevoflurane, whereas larger amounts of reaction products were observed with sodium hydroxide and potassium hydroxide. The alkali hydroxides of sodalime are presumably responsible for its reaction with halogenated inhalation anaesthetics. We therefore conclude that decomposing reactions of halogenated inhalation anesthetics with dry soda lime could be prevented by using a newly developed soda lime.

[Elimination of hydroxyethyl starch after autologous retransfusion of cryopreserved erythrocytes]. / Elimination von Hydroxyethylstärke nach autologer Retransfusion von damit kryokonservierten Erythrozyten.

In the case of the biodegradable cryoprotectant hydroxyethyl starch (HES) no deglycerolization process is required prior to the transfusion of frozen red blood cells (RBC). In a first study the elimination of an HES 200,000/0.62 from the plasma of 6 dogs was investigated by means of a novel HPLC-GFC method. 16% of the blood volume were replaced by autologous HES protected frozen/thawed RBC. In a second study the HES concentration in the plasma of 7 healthy volunteers was determined following the substitution of 8% of the blood volume, but a washing step has been performed to reduce the concentration of the cryoprotectant (HES 200,000/0.5). In a third study, however, this step was omitted. The elimination of the HES followed always first order kinetics. In the case of the transfusions without postthaw washing in dogs and humans, the initial plasma concentrations amounted to 2.11 +/- 0.15 g/dl and 0.75 +/- 0.26 g/dl, respectively. The corresponding value for the washed preparations was less than 0.03 g/dl. Within 4-5 h the concentrations dropped to less than 50% of the initial values. The 9-hour value was less than 35% (dogs), the 20-hour value about 15% (humans) of the initial concentration. As HES is primarily eliminated via the kidneys, within this period the concentrations of HES in the urine dropped from 4.3 +/- 2.11 g/dl to less than 0.03 g/dl (humans, no washing step). In conclusion, the elimination of the accompanying cryoprotectant HES was no problem in the concentrations applied. A simple washing step with isotonic saline, however, effectively reduced the concentration of the extracellular cryoprotectant HES far below critical levels.