The coordination of copper(II) with β-casomorphin and its fragments

The synthesis of β-casomorphin-5 (Tyr-Pro-Phe-Pro-Gly, H₂L) and a number of its peptide fragments is described. Complexes formed between these peptides and Cu(II) have been investigated spectrophotometrically, using CD and EPR spectroscopy, and potentiometrically. Results show that, with tyrosine as the N-terminal residue, the major complex formed at physiological pH is the dimeric species, [Cu₂L₂], bonded through the phenolic O^? of the Tyr residue of one ligand and the N-terminal amine nitrogen of the second ligand molecule. There is no evidence for coordination through the peptide nitrogens unless the terminal Tyr group is removed.     

Loss of CDK5RAP2 affects neural but not non-neural mESC differentiation into cardiomyocytes

Biallelic mutations in the gene encoding centrosomal CDK5RAP2 lead to autosomal recessive primary microcephaly (MCPH), a disorder characterized by pronounced reduction in volume of otherwise architectonical normal brains and intellectual deficit. The current model for the microcephaly phenotype in MCPH invokes a premature shift from symmetric to asymmetric neural progenitor-cell divisions with a subsequent depletion of the progenitor pool. The isolated neural phenotype, despite the ubiquitous expression of CDK5RAP2, and reports of progressive microcephaly in individual MCPH cases prompted us to investigate neural and non-neural differentiation of Cdk5rap2-depleted and control murine embryonic stem cells (mESC). We demonstrate an accumulating proliferation defect of neurally differentiating Cdk5rap2-depleted mESC and cell death of proliferative and early postmitotic cells. A similar effect does not occur in non-neural differentiation into beating cardiomyocytes, which is in line with the lack of non-central nervous system features in MCPH patients. Our data suggest that MCPH is not only caused by premature differentiation of progenitors, but also by reduced propagation and survival of neural progenitors.     

Is it possible to orally vaccinate juvenile red foxes against rabies in spring campaigns?

The rabies antibody status of juvenile foxes (Vulpes vulpes) was evaluated in large-scale, long-term oral vaccination campaigns. Between 9% (n = 659) and 21% (n = 42) of the juvenile foxes examined in 1993-94 and 1997, respectively, showed rabies virus neutralizing antibody (nAb)-titers > or = 0.5 IU/ml following bait distribution in spring. The presence of nAb may be due to either the passive transfer of maternal antibodies, or active immunization derived from spring vaccination campaigns. The latter alternative is supported by the finding of nAb throughout late spring and the summer months, and the finding of the tetracycline (TC) biomarker, used in the vaccine-baits, in 27% (n = 43) and 37% (n = 155) of juveniles in 1993-94 and 1997, respectively. It was not possible to distinguish nAb originating from passive immunity from that arising from active immunization. However, biological data on the whelping period of red foxes, on dynamics of maternal antibodies and the timing of oral vaccination, gave evidence that a superposition of these processes is likely. Evidence from these studies suggests that oral vaccination coinciding with the spring perinatal period may produce immunity in both parents and only in a certain percentage of the offspring simultaneously. This phenomenon should be useful in further enhancing the efficacy of oral vaccination in red foxes.     

Potent bradykinin antagonists containing N-benzylglycine or N-benzyl-l-alanine in position 8.

Two new analogues of a previously designed bradykinin (BK) antagonist, d-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-d-Phe-Thi-Arg, substituted in position 8 by N-benzylglycine and N-benzyl-l-alanine were designed, synthesized and bioassayed. The results show an impressive enhancement of B2 antagonistic potencies of both peptides in comparison with the model. In two further analogues these modifications were combined with acylation of the N-terminus with 1-adamantanacarboxylic acid. Acylated analogues exhibited higher antagonistic potency in comparison with the parent compounds, however, the range of effect was not as high as in previously described cases. The activity of analogues was assessed by their ability to inhibit vasodepressor response to exogenous BK (rat blood pressure test). Our results may be of value in the design of more potent BK antagonists.     

Saddle-Point Approximations,Integrodifference Equations,and Invasions

Invasion, the growth in numbers and spatial spread of a population over time, is a fundamental process in ecology. Governments and businesses expend vast sums to prevent and control invasions of pests and pestilences and to promote invasions of endangered species and biological control agents. Many mathematical models of biological invasions use nonlinear integrodifference equations to describe the growth and dispersal processes and to predict the speed of invasion fronts. Linear models have received less attention, perhaps because they are difficult to simulate for large times. In this paper, we use the saddle-point method, alias the method of steepest descent, to derive asymptotic approximations for the solutions of linear integrodifference equations. We work through five examples, for Gaussian, Laplace, and uniform dispersal kernels in one dimension and for asymmetric Gaussian and radially symmetric Laplace kernels in two dimensions. Our approximations are extremely close to the exact solutions, even for intermediate times. We also employ an empirical saddle-point approximation to predict densities using dispersal data. We use our approximations to examine the effects of censored dispersal data on estimates of invasion speed and population density.     

Proteasome inhibition drastically but reversibly impairs murine lymphocyte development

The proteasome inhibitor bortezomib, which induces cell death in various cancer cell lines including lymphatic neoplasias, has recently been approved for the treatment of relapsed multiple myeloma. Important mechanisms of proteasome inhibitor-mediated tumor cell death are the inhibition of NF-kappaB activation and induction of the terminal unfolded protein response (UPR). However, little is known about effects of bortezomib on developing and mature lymphocytes. Therefore, Balb/C mice were injected with bortezomib and lymphocyte subsets were analyzed. This treatment resulted in dramatically decreased numbers of T and B lymphocyte precursors, while mature lymphocytes were only partially affected. Thymocytes were almost depleted 3 days after a single bortezomib injection, pro-B and pre-B cells already after 2 days. Thymocytes and B cell precursors recovered within 2 weeks. The decreased numbers of developing lymphocytes were due to apoptotic cell death accompanied by strongly increased caspase 3/7 activity. Within 8 h after bortezomib injection, there was a strong induction of heat shock protein 70 and C/EBP homologous protein in bone marrow B cells, indicating endoplasmic reticulum stress and activation of the terminal UPR, respectively. Hence, induction of apoptosis by proteasome inhibition can dramatically affect lymphocyte development, a fact which has important implications for the clinical use of bortezomib, especially in situations with ongoing lymphopoiesis.     

ABRF-PRG05: De Novo Peptide Sequence Determination

A common request of proteomics core facilities is protein identification. However, in some instances primary sequence information for the protein in question is not present in public databases. In other cases, the amino acid sequence of a protein may differ in some way from the sequence predicted from the gene sequence in a database as a result of gene mutation, gene splicing, and/or multiple posttranslational modifications. Thus, it may be necessary to determine the sequence of one or more peptides de novo in order to identify and/or adequately characterize the protein of interest. The primary goal of this study was to give participating laboratories an opportunity to evaluate their proficiency in sequencing unknown peptides that are not included in any published database. Samples containing 3–6 pmol each of five synthetic peptides with amino acid sequences that were not present in public databases were sent to 106 laboratories. One nonstandard amino acid was present in one of the peptides. From a comparison of the results obtained by different strategies, participating laboratories will be able to gauge their own capabilities and establish realistic expectations for the approaches that can be used for this determination.     

Radiocarbon based assessment of soil organic matter contribution to soil respiration in a pine stand of the Campine region, Belgium

The contribution of decomposing soil organic carbon (SOC) to total annual soil respiration (SR) was evaluated by radiocarbon measurements at a Scots pine stand growing on a plaggen soil in the Belgian Campine region. Two approaches were used to estimate the contribution of different C pools to SR. In the first approach, the variations in ¹⁴C content of soil CO₂ efflux were monitored during one year (2003) and compared to the atmospheric and SOC ¹⁴C signatures to determine the contribution of ??fast?? (root respiration and fast decomposing SOC) and ??slow?? cycling C pools to total SR. In the second approach an estimate of the total heterotrophic soil respiration (Rh), comprising the slow cycling C and the heterotrophic part of the fast-cycling C pools, was derived applying a box model based on the amount of the bulk SOC pool and its ¹⁴C-derived mean residence time (MRT). The quantification of the Rh and the decomposition rate of the slow-cycling SOC allows to indirectly determining the contribution of the heterotrophic C that decompose within a year. Measurements of total SR performed in the field allowed assessing the contribution of the different C pools to total soil C efflux. On an annual basis, the fast-cycling C was the main contributor to SR, about 85%, while the contribution of the slow-cycling C (with MRT >1 yr) to total SR was 15%. Total annual Rh was 36% of total SR, which is in the lower range reported for temperate coniferous forests. The comparison of Rh with other estimates for the same site (47?C50% of total SR) suggest a possible underestimation of the C flux from the mineral soil. In fact, the ??very old?? C contained in the plaggen horizon strongly affects the signature of the mostly young C leaving the soil. In conclusion, our results indicate that the contribution of SOC decomposition to total soil CO₂ flux in this forest is less than 40%, and at least half of it comes from organic compounds less than 1 year old.