Adjuvant low-dose interferon α2a with or without dacarbazine compared with surgery alone: a prospective-randomized phase III DeCOG trial in melanoma patients with regional lymph node metastasis

Background: More than half of patients with melanoma that has spread to regional lymph nodes develop recurrent disease within the first 3 years after surgery. The aim of the study was to improve disease-free survival (DFS) and overall survival (OS) with interferon (IFN) α2a with or without dacarbazine (DTIC) compared with observation alone. Patients and methods: A total of 444 patients from 42 centers of the German Dermatologic Cooperative Oncology Group who had received a complete lymph node dissection for pathologically proven regional node involvement were randomized to receive either 3 MU s.c. of IFNα2a three times a week for 2 years (Arm A) or combined treatment with same doses of IFNα2a plus DTIC 850 mg/m2 every 4-8 weeks for 2 years (Arm B) or to observation alone (Arm C). Treatment was discontinued at first sign of relapse. Results: A total of 441 patients were eligible for intention-to-treat analysis. Kaplan-Meier 4-year OS rate of those who had received IFNα2a was 59%. For those with surgery alone, survival was 42% (A versus C, P = 0.0045). No improvement of survival was found for the combined treatment Arm B with 45% survival rate (B versus C, P = 0.76). Similarly, DFS rates showed significant benefit for Arm A, and not for Arm B. Multivariate Cox model confirmed that Arm A has an impact on OS (P = 0.005) but not Arm B (P = 0.34). Conclusions: 3 MU interferon α2a given s.c. three times a week for 2 years significantly improved OS and DFS in patients with melanoma that had spread to the regional lymph nodes. Interestingly, the addition of DTIC reversed the beneficial effect of adjuvant interferon α2a therap

Dacarbazine and interferon α with or without interleukin 2 in metastatic melanoma: a randomized phase III multicentre trial of the Dermatologic Cooperative Oncology Group (DeCOG)

In several phase II-trials encouraging tumour responses rates in advanced metastatic melanoma (stage IV; AJCC-classification) have been reported for the application of biochemotherapy containing interleukin 2. This study was designed to compare the efficacy of therapy with dacarbazine (DTIC) and interferon α (IFN-α) only to that of therapy with DTIC and IFN-α with the addition of interleukin 2 (IL-2) in terms of the overall survival time and rate of objective remissions and to provide an elaborated toxicity profile for both types of therapy. 290 patients were randomized to receive either DTIC (850 mg/m2every 28 days) plus IFN-α2a/b (3 MIU/m2, twice on day 1, once daily from days 2 to 5; 5 MIU/m23 times a week from week 2 to 4) with or without IL-2 (4.5 MIU/m2for 3 hours i.v. on day 3; 9.0 MIU/m2i.v. day 3/4; 4.5 MIU/m2s.c. days 4 to 7). The treatment plan required at least 2 treatment cycles (8 weeks of therapy) for every patient. Of 290 randomized patients 281 were eligible for an intention-to-treat analysis. There was no difference in terms of survival time from treatment onset between the two arms (median 11.0 months each). In 273 patients treated according to protocol tumour response was assessable. The response rates did not differ between both arms (P = 0.87) with 18.0% objective responses (9.7% PR; 8.3% CR) for DTIC plus IFN-α as compared to 16.1% (8.8% PR; 7.3% CR) for DTIC, IFN-α and IL-2. Treatment cessation due to adverse reactions was significantly more common in patients receiving IL-2 (13.9%) than in patients receiving DTIC/IFN-α only (5.6%). In conclusion, there was neither a difference in survival time nor in tumour response rates when IL-2, applied according to the combined intravenous and subcutaneous schedule used for this study, was added to DTIC and IFN-α. However, toxicity was increased in melanoma patients treated with IL-2. Further phase III trials with continuous infusion and higher dosages must be performed before any final conclusions can be drawn on the potential usefulness of IL-2 in biochemotherapy of advanced melanoma. © 2001 Cancer Research Campaign http://www.bjcancer.co

Der Film "Ewiger Wald" - oder: Die Überwindung der Zeit durch den Raum. Eine filmische Umsetzung von Rosenbergs "Mythus des 20. Jahrhunderts"

Der Autor erörtert den Film „Ewiger Wald“ (1936), welcher zu den bisher kaum erforschten nationalsozialistischen Experimentierfilmen gehört und die radikale Weltanschauung im Sinne des „Mythus des 20. Jahrhunderts" unmittelbar in Kunst fürs Volk umsetzen wollte. Er geht auf die Auftraggeber und Gestalter ein und erläutert eine neue ästhetische Produktion: „Filmdichtung", und die Symbolik des Deutsches Wald-Volk und die "Wald"-Moral, um dann auf den heroischen Mythos und völkische Rituale einzugehen. Anschließend erklärt er warum es sich auch um einen radikalisierten Umweltschutzfilm von Rechts handelt und welche politischen Wirkungsabsichten gegeben waren. Abschließend klärt er die Frage, ob es sich um einen erfolgreichen Film handelt. (DIPF/Orig./ah

Site-Specific Protonation Kinetics of Acidic Side Chains in Proteins Determined by pH-Dependent Carboxyl (13)C NMR Relaxation.

Proton-transfer dynamics plays a critical role in many biochemical processes, such as proton pumping across membranes and enzyme catalysis. The large majority of enzymes utilize acid-base catalysis and proton-transfer mechanisms, where the rates of proton transfer can be rate limiting for the overall reaction. However, measurement of proton-exchange kinetics for individual side-chain carboxyl groups in proteins has been achieved in only a handful of cases, which typically have involved comparative analysis of mutant proteins in the context of reaction network modeling. Here we describe an approach to determine site-specific protonation and deprotonation rate constants (kon and koff, respectively) of carboxyl side chains, based on (13)C NMR relaxation measurements as a function of pH. We validated the method using an extensively studied model system, the B1 domain of protein G, for which we measured rate constants koff in the range (0.1-3) × 10(6) s(-1) and kon in the range (0.6-300) × 10(9) M(-1) s(-1), which correspond to acid-base equilibrium dissociation constants (Ka) in excellent agreement with previous results determined by chemical shift titrations. Our results further reveal a linear free-energy relationship between log kon and pKa, which provides information on the free-energy landscape of the protonation reaction, showing that the variability among residues in these parameters arises primarily from the extent of charge stabilization of the deprotonated state by the protein environment. We find that side-chain carboxyls with extreme values of koff or kon are involved in hydrogen bonding, thus providing a mechanistic explanation for the observed stabilization of the protonated or deprotonated state

Ganglioside lipids accelerate α-synuclein amyloid formation

The deposition of α-synuclein fibrils is one hallmark of Parkinson's disease. Here, we investigate how ganglioside lipids, present in high amounts in neurons and exosomes, influence the aggregation kinetics of α-synuclein. Gangliosides, as well as, other anionic lipid species with small or large headgroups were found to induce conformational changes of α-synuclein monomers and catalyse their aggregation at mildly acidic conditions. Although the extent of this catalytic effect was slightly higher for gangliosides, the results imply that charge interactions are more important than headgroup chemistry in triggering aggregation. In support of this idea, uncharged lipids with large headgroups were not found to induce any conformational change and only weakly catalyse aggregation. Intriguingly, aggregation was also triggered by free ganglioside headgroups, while these caused no conformational change of α-synuclein monomers. Our data reveal that partially folded α-synuclein helical intermediates are not required species in triggering of α-synuclein aggregation

The A beta 40 and A beta 42 peptides self-assemble into separate homomolecular fibrils in binary mixtures but cross-react during primary nucleation

The assembly of proteins into amyloid fibrils, a phenomenon central to several currently incurable human diseases, is a process of high specificity that commonly tolerates only a low level of sequence mismatch in the component polypeptides. However, in many cases aggregation-prone polypeptides exist as mixtures with variations in sequence length or post-translational modifications; in particular amyloid beta (A beta) peptides of variable length coexist in the central nervous system and possess a propensity to aggregate in Alzheimer's disease and related dementias. Here we have probed the co-aggregation and cross-seeding behavior of the two principal forms of A beta, A beta 40 and A beta 42 that differ by two hydrophobic residues at the C-terminus. We find, using isotope-labeling, mass spectrometry and electron microscopy that they separate preferentially into homomolecular pure A beta 42 and A beta 40 structures during fibril formation from mixed solutions of both peptides. Although mixed fibrils are not formed, the kinetics of amyloid formation of one peptide is affected by the presence of the other form. In particular monomeric A beta 42 accelerates strongly the aggregation of A beta 40 in a concentration-dependent manner. Whereas the aggregation of each peptide is catalyzed by low concentrations of preformed fibrils of the same peptide, we observe a comparably insignificant effect when A beta 42 fibrils are added to A beta 40 monomer or vice versa. Therefore we conclude that fibril-catalysed nucleus formation and elongation are highly sequence specific events but A beta 40 and A beta 42 interact during primary nucleation. These results provide a molecular level description of homomolecular and heteromolecular aggregation steps in mixtures of polypeptide sequence variants

A Microfluidic Platform for Real-Time Detection and Quantification of Protein-Ligand Interactions

The key steps in cellular signaling and regulatory pathways rely on reversible noncovalent protein-ligand binding, yet the equilibrium parameters for such events remain challenging to characterize and quantify in solution. Here, we demonstrate a microfluidic platform for the detection of protein-ligand interactions with an assay time on the second timescale and without the requirement for immobilization or the presence of a highly viscous matrix. Using this approach, we obtain absolute values for the electrophoretic mobilities characterizing solvated proteins and demonstrate quantitative comparison of results obtained under different solution conditions. We apply this strategy to characterize the interaction between calmodulin and creatine kinase, which we identify as a novel calmodulin target. Moreover, we explore the differential calcium ion dependence of calmodulin ligand-binding affinities, a system at the focal point of calcium-mediated cellular signaling pathways. We further explore the effect of calmodulin on creatine kinase activity and show that it is increased by the interaction between the two proteins. These findings demonstrate the potential of quantitative microfluidic techniques to characterize binding equilibria between biomolecules under native solution conditions

Ultrastructural evidence for self-replication of alzheimer-associated Aβ42 amyloid along the sides of fibrils

The nucleation of Alzheimer-associated Aβ peptide monomers can be catalyzed by preexisting Aβ fibrils. This leads to autocatalytic amplification of aggregate mass and underlies self-replication and generation of toxic oligomers associated with several neurodegenerative diseases. However, the nature of the interactions between the monomeric species and the fibrils during this key process, and indeed the ultrastructural localization of the interaction sites have remained elusive. Here we used NMR and optical spectroscopy to identify conditions that enable the capture of transient species during the aggregation and secondary nucleation of the Aβ42 peptide. Cryo-electron microscopy (cryo-EM) images show that new aggregates protrude from the entire length of the progenitor fibril. These protrusions are morphologically distinct from the wellordered fibrils dominating at the end of the aggregation process. The data provide direct evidence that self-replication through secondary nucleation occurs along the sides of fibrils, which become heavily decorated under the current solution conditions (14 μM Aβ42, 20 mM sodium phosphate, 200 μM EDTA, pH 6.8)