Anchor-linked intermediates in peptide amide synthesis are caused by dimeric anchors on the solid supports

Cleavage and kinetic studies have been carried out using commercially obtained H-Tyr(tBu)-5-(4′-aminomethyl-3′,5′-dimethoxyphenoxy)valeric acid-TentaGelS (H-Tyr(tBu)-4-ADPV-TentaGelS) and H-Tyr (tBu)-4-ADPV-Ala-aminomethyl-resin (H-Tyr(tBu)-4-ADPV-AM-resin) prepared from commercially available resin and loaded with commercially available Fmoc-4-ADPV-OH amide anchor. Cleavage with pure trifluoroacetic acid (TFA) gave the intermediate H-Tyr-4-ADPV-NH₂, which was then degraded to H-Tyr-NH₂, and cleavage with TFA/dichloromethane (1:9) yielded H-Tyr-4-ADPV-NH₂ which could be isolated in preparative amounts. Cleavage reactions with ¹⁵N-labelled H-Ala-4-ADPV-[¹⁵N]-Gly-AM-resin yielded the intermediate H-Ala-4-ADPV-NH₂, which contained no ¹⁵N as demonstrated by ¹H-NMR. The analysis of the commercial Fmoc-4-ADPV-OH amide anchor showed the presence of Fmoc-4-ADPV-4-ADPV-OH as an impurity in high amounts. This dimeric anchor molecule is the cause of formation of the anchor-linked peptide intermediate obtained during the cleavage from the resin. The particularly high acid-lability of the amide bond between the two ADPV moieties was utilized to synthesize sidechain and C-terminally 4-ADPV protected pentagastrin on a double-anchor resin, and to cleave it using 5% trifluoroacetic acid in dichloromethane. This method may offer a new way for the synthesis of protected peptide amides with improved solubility to be used in fragment condensation.     

G2 phase cell cycle disturbance as a manifestation of genetic cell damage

The predominant cell cycle change induced by X-rays and clastogens in peripheral blood mononuclear cells is the accumulation of cells in the G2 phase of the cell cycle. We show that this accumulation consists of cells that are either delayed or arrested within the G2 phase. Since both X-rays and DNA crosslinking chemicals are known to damage DNA, the G2 phase inhibition caused by these agents is thought to be one of the primary manifestations of (unrepaired) DNA damage. This interpretation is supported by two additional findings. (1) Older individuals have elevated baseline levels of mononuclear blood cells that are delayed and/or arrested in the G2 phase of the cell cycle. This coincides with the increased chromosomal breakage rates reported for older individuals. (2) Irrespective of their age, individuals with inherited genetic instability syndromes (such as Fanconi anemia and Bloom syndrome) exhibit elevated G2 phase cell fractions. We show that the method used to detect such induced or spontaneous cell cycle changes, viz. BrdU-Hoechst flow cytometry, is a rapid and highly sensitive technique for the assessment of genetic cell damage.Dedicated to Professor Ulrich Wolf on the occasion of his 60th birthday     

B-cells of the synovial membrane. II. Differentiation during development of the synovial cavity in the mouse

Summary Study of pre- and postnatal development of the metatarsophalangeal joint of the mouse shows that the synovial cavity (SC) forms before any differentiation of the synovial mesenchyme. The primitive cleft results from degradation of a thin vascular mesenchymal layer in direct contact with the chondrogenic layers. Differentiation of the synovial membrane coincides with clarification of the SC (3rd to 6th day of postnatal life). When dilatation of the SC occurs (6th to 8th day), the two intimal cells types (A- and B-cells) are well identified. The B-cells already show typical features at day 6; their content of typical dense secretory vesicles is comparable to that of the adult B-cells at day 13. The specific secretory function of B-cells could be correlated with the particular structure of the intimal interstitial tissue and could account for the origin of some protein(s) of the synovial fluid.ERA 178 (Neuroendocrinologie Comparée) du CNRS et INSERM     

Microbial Activities in Undecompressed and Decompressed Deep-Seawater Samples

Microbial transformations of ¹⁴C-labeled substrates (sodium glutamate, Casamino Acids, glucose, and sodium acetate) were measured in undecompressed seawater samples collected from depths of 1,800 to 6,000 m, during 14- to 21-day incubation periods at in situ temperature (3°C). Each substrate was tested at two concentrations (ca. 0.5 and 5.0 μg/ml) and two in situ pressures. The data were compared to 1-atmosphere (ca. 1.013 × 10² kPa) controls. The rates of ¹⁴C incorporation and ¹⁴CO₂ production as well as the amounts of total substrate utilization were generally lower at pressure than in the decompressed controls but were significantly different for each of the four substrates used. The utilization of acetate was the least affected by pressure; rates were similar to those measured at 1 atmosphere in two out of four experiments. In contrast, transformation rates of the amino acids at pressure averaged to only 38% of those in the controls. A single but reproducible “barophilic” response was observed with glucose as a substrate in samples collected from a depth of 4,500 m at a specific area in the northwestern Atlantic Ocean. Except for this latter set of experiments, the transformation of all substrates showed an increased lag period at pressure as compared to the 1-atmosphere controls.     

The impact of mtDNA analysis between positions nt8306 and nt9021 for forensic casework

Identification in forensic casework by mtDNA sequencing is predominantly done by sequencing the non-coding control region (HVI and HVII). In an attempt to further increase the discriminatory power of mtDNA analysis, we sequenced a coding region between nt8306 and nt9021 to identify additional polymorphisms in a group of 61 unrelated German individuals who had mtDNA profiles that occurred more than two times each, as well as a control group of 119 unrelated Germans whose profiles occurred one or two times each. Within these 180 individuals, 38 different polymorphisms in this region were observed; 64.4% of these individuals displayed the Cambridge reference sequence profile plus A8860G. For 28 individuals with the two most common profiles, A263G-315.1insC (N = 18) and A263G-309.1insC-315.1insC (N = 10), additional polymorphisms in this coding region permitted further discrimination of 56 and 40% of the individuals, respectively.     

Suppression of Electron Transfer to Dioxygen by Charge Transfer and Electron Transfer Complexes in the FAD-dependent Reductase Component of Toluene Dioxygenase

The three-component toluene dioxygenase system consists of an FAD-containing reductase, a Rieske-type [2Fe-2S] ferredoxin, and a Rieske-type dioxygenase. The task of the FAD-containing reductase is to shuttle electrons from NADH to the ferredoxin, a reaction the enzyme has to catalyze in the presence of dioxygen. We investigated the kinetics of the reductase in the reductive and oxidative half-reaction and detected a stable charge transfer complex between the reduced reductase and NAD⁺ at the end of the reductive half-reaction, which is substantially less reactive toward dioxygen than the reduced reductase in the absence of NAD⁺. A plausible reason for the low reactivity toward dioxygen is revealed by the crystal structure of the complex between NAD⁺ and reduced reductase, which shows that the nicotinamide ring and the protein matrix shield the reactive C4a position of the isoalloxazine ring and force the tricycle into an atypical planar conformation, both factors disfavoring the reaction of the reduced flavin with dioxygen. A rapid electron transfer from the charge transfer complex to electron acceptors further reduces the risk of unwanted side reactions, and the crystal structure of a complex between the reductase and its cognate ferredoxin shows a short distance between the electron-donating and -accepting cofactors. Attraction between the two proteins is likely mediated by opposite charges at one large patch of the complex interface. The stability, specificity, and reactivity of the observed charge transfer and electron transfer complexes are thought to prevent the reaction of reductase_TOL with dioxygen and thus present a solution toward conflicting requirements.     

Cardiac Per2 Functions as Novel Link between Fatty Acid Metabolism and Myocardial Inflammation during Ischemia and Reperfusion Injury of the Heart

Disruption of peripheral circadian rhyme pathways dominantly leads to metabolic disorders. Studies on circadian rhythm proteins in the heart indicated a role for Clock or Per2 in cardiac metabolism. In contrast to Clock^−/−, Per2^−/− mice have larger infarct sizes with deficient lactate production during myocardial ischemia. To test the hypothesis that cardiac Per2 represents an important regulator of cardiac metabolism during myocardial ischemia, we measured lactate during reperfusion in Per1^−/−, Per2^−/− or wildtype mice. As lactate measurements in whole blood indicated an exclusive role of Per2 in controlling lactate production during myocardial ischemia, we next performed gene array studies using various ischemia-reperfusion protocols comparing wildtype and Per2^−/− mice. Surprisingly, high-throughput gene array analysis revealed dominantly lipid metabolism as the differentially regulated pathway in wildtype mice when compared to Per2^−/−. In all ischemia-reperfusion protocols used, the enzyme enoyl-CoA hydratase, which is essential in fatty acid beta-oxidation, was regulated in wildtype animals only. Studies using nuclear magnet resonance imaging (NMRI) confirmed altered fatty acid populations with higher mono-unsaturated fatty acid levels in hearts from Per2^−/− mice. Unexpectedly, studies on gene regulation during reperfusion revealed solely pro inflammatory genes as differentially regulated ‘Per2-genes’. Subsequent studies on inflammatory markers showed increasing IL-6 or TNFα levels during reperfusion in Per2^−/− mice. In summary, these studies reveal an important role of cardiac Per2 for fatty acid metabolism and inflammation during myocardial ischemia and reperfusion, respectively.