Cyclic AMP-induced reversible decrease in cAMP-binding to cell surface receptors of Dictyostelium discoideum

Abstract Adaptation may be the result of a change in affinity and/or number of cAMP-binding sites at the cell surface. To test this possibility we used agip 53, a mutant that does not synthesize cAMP in response to cAMP stimulation. cAMP induced a fast decrease in cAMP-binding to aggregation-competent cells, which reached a maximum at 10–20 s and was reversible with a t _0.5 of about 70 s. The decrease in cAMP-binding involved 46000 sites per cell and was mainly due to a reduction in the apparent affinity for cAMP-binding and to a smaller extent to slowly dissociating cAMP. Our results suggest that under these conditions only a fraction of the cAMP-binding sites at the cell surface are involved in transmembrane signalling, which is indeed observed for many of the physiological responses in Dictyostelium discoideum .     

Refining CLAMP — Investigations towards improving the Climate Leaf Analysis Multivariate Program

CLAMP (Climate Leaf Analysis Multivariate Program) has been used for the past 17 years to estimate palaeoclimatic conditions. The reliability and applicability of this method, based on leaf physiognomic characters of fossil woody dicots, has been widely discussed over the same period. The present study focuses on some technical aspects of CLAMP, mainly on its robustness in the context of the theoretical unimodal requirements of Canonical Correspondence Analysis, and introduces “correction coefficients” for these aspects of the statistical approach as a new way of interpreting and improving on CLAMP estimates. This tool was tested on datasets derived from 17 European fossil floras ranging in age from the Late Oligocene to the Pliocene. Additionally, an objective statistical method for the selection of the best-suited modern vegetation dataset from 144 site (Physg3br) or 173 (Physg3ar) extant biotopes is proposed.     

Reconstruction of pre-monsoon weather conditions in northwestern Thailand from the tree-ring widths of Pinus merkusii and Pinus kesiya

Two pine species, Pinus merkusii and Pinus kesiya, native in Thailand were studied for their potential to reconstruct past weather conditions from their tree rings. Altogether, cores from 209 Merkus pines and from 205 Khasi pines were sampled at 16 sites. Standard methods were applied to assemble tree-ring chronologies for each site and tree species. The longest site chronology of Merkus pine and Khasi pine covered 314 and 183 years, respectively. Principal component analysis (PCA) illustrated a close clustering of all, except two, site chronologies on the PC1 axis, indicating a strong signal common for both pine species throughout NW Thailand. However, along the PC2 axis, there was a distinct separation between the Merkus pine and the Khasi pine site chronologies, which was possibly due to the species-specific differences in site altitudes. Simple correlations between the PC1 time series and a regionalized climate data set showed a highly negative association with temperature during the pre-monsoon period from March to May. Since, pre-monsoon temperature and rainfall were negatively correlated with each other, we used the PC1 as a direct proxy for pre-monsoon weather conditions (warm/dry vs. cool/wet) back to 1834 AD.     

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.     

L1 is sequentially processed by two differently activated metalloproteases and presenilin/gamma-secretase and regulates neural cell adhesion, cell migration, and neurite outgrowth

The immunoglobulin superfamily recognition molecule L1 plays important functional roles in the developing and adult nervous system. Metalloprotease-mediated cleavage of this adhesion molecule has been shown to stimulate cellular migration and neurite outgrowth. We demonstrate here that L1 cleavage is mediated by two distinct members of the disintegrin and metalloprotease family, ADAM10 and ADAM17. This cleavage is differently regulated and leads to the generation of a membrane bound C-terminal fragment, which is further processed through gamma-secretase activity. Pharmacological approaches with two hydroxamate-based inhibitors with different preferences in blocking ADAM10 and ADAM17, as well as loss of function and gain of function studies in murine embryonic fibroblasts, showed that constitutive shedding of L1 is mediated by ADAM10 while phorbol ester stimulation or cholesterol depletion led to ADAM17-mediated L1 cleavage. In contrast, N-methyl-d-aspartate treatment of primary neurons stimulated ADAM10-mediated L1 shedding. Both proteases were able to affect L1-mediated adhesion and haptotactic migration of neuronal cells. In particular, both proteases were involved in L1-dependent neurite outgrowth of cerebellar neurons. Thus, our data identify ADAM10 and ADAM17 as differentially regulated L1 membrane sheddases, both critically affecting the physiological functions of this adhesion protein.     

A synthetic amino acid substitution of Tyr10 in Aβ peptide sequence yields a dominant negative variant in amyloidogenesis

Alzheimer's disease (AD) is the most common cause of dementia in elderly people, and age is the major nongenetic risk factor for sporadic AD. A hallmark of AD is the accumulation of amyloid in the brain, which is composed mainly of the amyloid beta-peptide (Aβ) in the form of oligomers and fibrils. However, how aging induces Aβ aggregation is not yet fully determined. Some residues in the Aβ sequence seem to promote Aβ-induced toxicity in association with age-dependent risk factors for AD, such as (i) increased GM1 brain membrane content, (ii) altered lipid domain in brain membrane, (iii) oxidative stress. However, the role of Aβ sequence in promoting aggregation following interaction with the plasma membrane is not yet demonstrated. As Tyr10 is implicated in the induction of oxidative stress and stabilization of Aβ aggregation, we substituted Tyr 10 with a synthetic amino acid that abolishes Aβ-induced oxidative stress and shows an accelerated interaction with GM1. This variant peptide shows impaired aggregation properties and increased affinity for GM1. It has a dominant negative effect on amyloidogenesis in vitro, in cellulo, and in isolated synaptosomes. The present study shed new light in the understanding of Aβ-membrane interactions in Aβ-induced neurotoxicity. It demonstrates the relevance of Aβ sequence in (i) Aβ-membrane interaction, underlining the role of age-dependent enhanced GM1 content in promoting Aβ aggregation, (ii) Aβ aggregation, and (iii) Aβ-induced oxidative stress. Our results open the way for the design of peptides aimed to inhibit Aβ aggregation and neurotoxicity.     

Proteomics-based target identification: bengamides as a new class of methionine aminopeptidase inhibitors

LAF389 is a synthetic analogue of bengamides, a class of marine natural products that produce inhibitory effects on tumor growth in vitro and in vivo. A proteomics-based approach has been used to identify signaling pathways affected by bengamides. LAF389 treatment of cells resulted in altered mobility of a subset of proteins on two-dimensional gel electrophoresis. Detailed analysis of one of the proteins, 14-3-3gamma, showed that bengamide treatment resulted in retention of the amino-terminal methionine, suggesting that bengamides directly or indirectly inhibited methionine aminopeptidases (MetAps). Both known MetAps are inhibited by LAF389. Short interfering RNA suppression of MetAp2 also altered amino-terminal processing of 14-3-3gamma. A high resolution structure of human MetAp2 co-crystallized with a bengamide shows that the compound binds in a manner that mimics peptide substrates. Additionally, the structure reveals that three key hydroxyl groups on the inhibitor coordinate the di-cobalt center in the enzyme active site.     

High-resolution peptide mapping of cerebrospinal fluid: a novel concept for diagnosis and research in central nervous system diseases

Peptides, such as many hormones, cytokines and growth factors play a central role in biological processes. Furthermore, as degradation products and processed forms of larger proteins they are part of the protein turnover. Thus, they can reflect disease-related changes in an organism's homeostasis in several ways. Since two-dimensional gel electrophoresis is restricted to analysis and display of proteins with relative molecular masses above 5000, we developed Differential Peptide Display (DPD), a new technology for analysis and visualization of peptides. Here we describe its application to cerebrospinal fluid of three subjects without a disease of the central nervous system (CNS) undergoing routine myelography and of two patients suffering from a primary CNS lymphoma. Peptides with a relative molecular mass below 20000 were extracted and analysed by a combination of chromatography and mass spectrometry. The peptide pattern of a sample was depicted as a multi-dimensional peptide mass fingerprint with each peptide's position being characterized by its molecular mass and chromatographic behaviour. Such a fingerprint of a CNS sample consists of more than 6000 different signals. Data analysis of peptide patterns from patients with CNS lymphoma compared to controls revealed obvious differences regarding the peptide content of the samples. By analysing peptides within a mass range of 750-20000, DPD extends 2D gel electrophoresis, thus offering the chance to investigate CNS diseases on the level of peptides. This represents a new approach for diagnosis and possible therapy.     

Model Construction and Analysis of Respiration in Halobacterium salinarum

The archaeon Halobacterium salinarum can produce energy using three different processes, namely photosynthesis, oxidative phosphorylation and fermentation of arginine, and is thus a model organism in bioenergetics. Compared to its bacteriorhodopsin-driven photosynthesis, less attention has been devoted to modeling its respiratory pathway. We created a system of ordinary differential equations that models its oxidative phosphorylation. The model consists of the electron transport chain, the ATP synthase, the potassium uniport and the sodium-proton antiport. By fitting the model parameters to experimental data, we show that the model can explain data on proton motive force generation, ATP production, and the charge balancing of ions between the sodium-proton antiporter and the potassium uniport. We performed sensitivity analysis of the model parameters to determine how the model will respond to perturbations in parameter values. The model and the parameters we derived provide a resource that can be used for analytical studies of the bioenergetics of H. salinarum.