New variants of inducible Cre recombinase: a novel mutant of Cre–PR fusion protein exhibits enhanced sensitivity and an expanded range of inducibility

We have developed a novel inducible Cre mutant with enhanced recombinase activity to mediate genetic switching events. The protein, designated Cre*PR, is composed of a new Cre mutant at the N-terminus followed by the ligand-binding domain (LBD) of the progesterone receptor (PR). The response to low doses of inducer is significantly enhanced by elongating the C-terminus of the PR LBD from amino acid 891 to 914. The mutant Cre lacks the first 18 amino acids and contains a Val→Ala substitution at position 336, thereby destroying a cryptic splice donor at the 3′-end of Cre. The latter mutation reduces unwanted background recombinase activity in the absence of the synthetic ligand RU486 by a factor of at least 10 to an almost undetectable level. Thus, the recombinase activity turns out to be inducible by a factor of >200. We expect Cre*PR to serve as a valuable tool for conditional expression of genes both in vitro and in vivo.     

New variants of inducible Cre recombinase: a novel mutant of Cre-PR fusion protein exhibits enhanced sensitivity and an expanded range of inducibility

We have developed a novel inducible Cre mutant with enhanced recombinase activity to mediate genetic switching events. The protein, designated Cre*PR, is composed of a new Cre mutant at the N-terminus followed by the ligand-binding domain (LBD) of the progesterone receptor (PR). The response to low doses of inducer is significantly enhanced by elongating the C-terminus of the PR LBD from amino acid 891 to 914. The mutant Cre lacks the first 18 amino acids and contains a Val-->Ala substitution at position 336, thereby destroying a cryptic splice donor at the 3'-end of CRE: The latter mutation reduces unwanted background recombinase activity in the absence of the synthetic ligand RU486 by a factor of at least 10 to an almost undetectable level. Thus, the recombinase activity turns out to be inducible by a factor of >200. We expect Cre*PR to serve as a valuable tool for conditional expression of genes both in vitro and in vivo.     

Identification of Ceramide Binding Proteins in Neuronal Cells: A Critical Point of View

Much discussion has centered on the biochemical mechanism by which ceramide is produced and functions as a signalling molecule in cells. To identify proteins involved in ceramide signalling, we synthesized a radioactively labelled ceramide analogue equipped with a photosensitive group: N-(p-trifluoromethyl-diazirinyl)phenyl-ethyl-2-[³⁵S]-2-thioacetyl-d-erythro-C₁₈-sphingosine ([³⁵S]-TDS-ceramide). This compound was then employed in photo-affinity labelling experiments in primary cultured cerebellar neurons. Due to the hydrophobic nature of the compound, most of the cell-associated radioactivity was recovered in the lipid fraction while only about 0.1% of radioactivity was photocoupled to proteins. In order to improve protein labelling the cytosolic fraction of rapidly growing human neuroblastoma cells (SH-SY5Y) was isolated and subjected to ceramide affinity chromatography prior to photo-affinity labelling. Following electrophoresis proteins photocoupled to ceramide were identified by MALDI mass spectrometry in combination with tryptic digestion and turned out to be either cytoskeletal or stress proteins that are highly abundant in cytosol and contain at least one hydrophobic domain.     

The kinetics of conformation change as determinant of Rubisco's specificity

The molecular basis of Rubisco's specificity is investigated in terms of the structure and kinetics of the enzyme. We propose that the rates of the conformational changes (closing/opening) of the binding niche exert a crucial influence on apparent binding rates and the enzyme's specificity. An extended reaction scheme for binding and conformational kinetics is presented and expressed in a mathematical model. The closed conformation, known from X-ray structures, is assumed to be necessary for binding of the gaseous substrates (carbon dioxide and oxygen) and for catalysis. Opening the niche interrupts catalysis and enables a fast exchange of those molecules between the internal cavity and the surrounding solvent. Our model predicts that specificity of Rubisco for CO₂ increases with the rate by which the niche opens. This is due to the fact that binding of the carbon dioxide is faster than oxygen binding, which is hampered by spin inversion. The apparent rate of carbon dioxide binding correlates with the repetition rate of the conformational change, and the rate of oxygen binding with the probability of the closed state. This revised version was published online in June 2006 with corrections to the Cover Date.     

Oxygen activation by isolated chloroplasts from Euglena gracilis. Ferredoxin-dependent function of a fluorescent compound and photosynthetic electron transport close to photosystem.

Oxygen reduction by isolated chloroplast lamellae from spinach, yielding the superoxide free radical in the light, is stimulated by a fluorescent factor (“compound No. 4”, isolated from Euglena gracilis strain Z) in a ferredoxin-dependent reaction. This reaction is not observed with Euglena chloroplasts, although there is a stimulation by compound No. 4 of ferredoxin-dependent oxygen reduction at the expense of NADPH + H⁺ as electron donor in the dark. Evidence is provided that in Euglena chloroplasts in the absence of NADP as electron acceptor a cyclic electron transport is predominating, including photosystem I, ferredoxin, NADP-ferredoxin reductase, and cytochrome₅₅₂. Isolated spinach chloroplast lamellae show a similar “cyclic” electron transport after treatment with digitonin, depending on the addition of the above cofactors. This result might indicate that Euglena chloroplast lamellae show this cyclic electron transport only as an artifact due to the isolation procedure. The results furthermore indicate that the pteridine-like, fluorescent compound No. 4 is not active as the primary electron acceptor of photosystem I; it may however be involved in oxygen activation by Euglena gracilis chloroplasts.     

Protein O-glycosylation analysis

Abstract This review provides an overview on the methods available for analysis of O-glycosylation. Three major themes are addressed: analysis of released O-glycans including different O-glycan liberation, derivatization, and detection methods; analysis of formerly O-glycosylated peptides yielding information on O-glycan attachment sites; analysis of O-glycopeptides, representing by far the most informative but also most challenging approach for O-glycan analysis. Although there are various techniques available for the identification of O-linked oligosaccharides, the focus here is on MS fragmentation techniques such as collision-induced fragmentation, electron capture dissociation, and electron transfer dissociation. Finally, the O-glycan analytical challenges that need to be met will be discussed.     

Interleukin-Encoding Adenoviral Vectors as Genetic Adjuvant for Vaccination against Retroviral Infection

Interleukins (IL) are cytokines with stimulatory and modulatory functions in the immune system. In this study, we have chosen interleukins which are involved in the enhancement of T_H2 responses and B cell functions to analyze their potential to improve a prophylactic adenovirus-based anti-retroviral vaccine with regard to antibody and virus-specific CD4⁺ T cell responses. Mice were vaccinated with an adenoviral vector which encodes and displays the Friend Virus (FV) surface envelope protein gp70 (Ad.pIXgp70) in combination with adenoviral vectors encoding the interleukins IL4, IL5, IL6, IL7 or IL23. Co-application of Ad.pIXgp70 with Ad.IL5, Ad.IL6 or Ad.IL23 resulted in improved protection with high control over FV-induced splenomegaly and reduced viral loads. Mice co-immunized with adenoviral vectors encoding IL5 or IL23 showed increased neutralizing antibody responses while mice co-immunized with Ad.IL6 or Ad.IL23 showed improved FV-specific CD4⁺ T cell responses compared to mice immunized with Ad.pIXgp70 alone. We show that the co-application of adenoviral vectors encoding specific interleukins is suitable to improve the vaccination efficacy of an anti-retroviral vaccine. Improved protection correlated with improved CD4⁺ T cell responses and especially with higher neutralizing antibody titers. The co-application of selected interleukin-encoding adenoviral vectors is a valuable tool for vaccination with regard to enhancement of antibody mediated immunity.     

Deficiency of Sphingosine-1-phosphate Lyase Impairs Lysosomal Metabolism of the Amyloid Precursor Protein

Progressive accumulation of the amyloid β protein in extracellular plaques is a neuropathological hallmark of Alzheimer disease. Amyloid β is generated during sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. In addition to the proteolytic processing by secretases, APP is also metabolized by lysosomal proteases. Here, we show that accumulation of intracellular sphingosine-1-phosphate (S1P) impairs the metabolism of APP. Cells lacking functional S1P-lyase, which degrades intracellular S1P, strongly accumulate full-length APP and its potentially amyloidogenic C-terminal fragments (CTFs) as compared with cells expressing the functional enzyme. By cell biological and biochemical methods, we demonstrate that intracellular inhibition of S1P-lyase impairs the degradation of APP and CTFs in lysosomal compartments and also decreases the activity of γ-secretase. Interestingly, the strong accumulation of APP and CTFs in S1P-lyase-deficient cells was reversed by selective mobilization of Ca²⁺ from the endoplasmic reticulum or lysosomes. Intracellular accumulation of S1P also impairs maturation of cathepsin D and degradation of Lamp-2, indicating a general impairment of lysosomal activity. Together, these data demonstrate that S1P-lyase plays a critical role in the regulation of lysosomal activity and the metabolism of APP.