Functional Characterization and Potential Applications for Enhanced Green Fluorescent Protein- and Epitope-Fused Human M1 Muscarinic Receptors

Four recombinant human M1 (hM1) muscarinic acetylcholine receptors (mAChRs) combining several modifications were designed and overexpressed in HEK293 cells. Three different fluorescent chimera were obtained through fusion of the receptor N terminus with enhanced green fluorescent protein (EGFP), potential glycosylation sites and a large part of the third intracellular (i3) loop were deleted, a hexahistidine tag sequence was introduced at the receptor C terminus, and, finally, a FLAG epitope was either fused at the receptor N terminus or inserted into its shortened i3 loop. High expression levels and ligand binding properties similar to those of the wild-type hM1 receptor together with confocal microscopy imaging demonstrated that the recombinant proteins were correctly folded and targeted to the plasma membrane, provided that a signal peptide was added to the N-terminal domain of the fusion proteins. Their functional properties were examined through McN-A-343-evoked Ca2+ release. Despite the numerous modifications introduced within the hM1 sequence, all receptors retained nearly normal abilities (EC50 values) to mediate the Ca2+ response, although reduced amplitudes (Emax values) were obtained for the i3-shortened constructs. Owing to the bright intrinsic fluorescence of the EGFP-fused receptors, their detection, quantitation, and visualization as well as the selection of cells with highest expression were straightforward. Moreover, the presence of the different epitopes was confirmed by immunocytochemistry. Altogether, this work demonstrates that these EGFP- and epitope-fused hM1 receptors are valuable tools for further functional, biochemical, and structural studies of muscarinic receptors.     

Isomerization between n-butyrate and isobutyrate in enrichment cultures

Abstract An isobutyrate-degrading methanogenic enrichment was obtained from a mesophilic anaerobic digester. Studies with growing cells and cell suspensions showed a reversible isomerization between butyrate and isobutyrate, suggesting that butyrate is an intermediate in the anaerobic degradation of isobutyrate. NMR experiments with ¹³C-labelled butyrate demonstrated that this isomerization resulted from the migration of the carboxyl group.     

Masking in Plants

Orcadian rhythms in plants are liable to masking, i.e. alterations by environmental influencing agents. Experiments have been reported for both positive and negative masking, attributed to a Zeitgeber which may either increase or decrease the amplitude of a circadian rhythm (CR). In some instances, the CR may even be unexpressed. This inhibition, however, may be alleviated by synchronizing agents. Reports are also available for changes in the shape or pattern of an oscillation. The latter may be prevented, at least in Acetabularia in certain conditions, by a phytohormone antagonist.

Masking may also be brought about by water stress, relative humidity, bacterial infection and alteration in the relative direction of the gravitational force.

Finally, subjecting plants to constant conditions, particularly continuous light, alters the physiological state of the organism.     

Implication of the First and Third Extracellular Loops of the μ-Opioid Receptor in the Formation of the Ligand Binding Site: A Study Using Chimeric μ-Opioid/Angiotensin Receptors

Abstract: Recent studies on chimeric μ/δ-, μ/κ- and δ/κ-opioid receptors have suggested that extracellular loops of the receptors were involved in the discriminatory binding of selective ligands by controlling their entry into the transmembrane binding site. Since homochimeric opioid receptors are mostly informative in terms of selectivity, the role of extracellular loops was examined here by studying heterochimeric μ receptors where the totality or parts of extracellular loops were replaced by the corresponding regions of the receptor for angiotensin II. Chimeric μ receptors with extracellular loop EL1 or EL3 originating from the angiotensin receptor had 100-fold decreased affinities for opioids; the length of the first extracellular loop, which is one residue longer in angiotensin than μ receptors, was shown to be responsible for this situation. Substitution of the μ receptor second extracellular loop by that of the angiotensin receptor diminished by ∼10-fold the affinities for opioids. Since all chimeras had altered affinities for selective and nonselective ligands, we propose that extracellular domains of the μ receptor, particularly the first and third loops, constrain the relative positioning of the connected transmembrane domains where selective as well as nonselective contact points form the opioid binding site.     

Characterization of substrate and product specificity of the purified recombinant glycogen branching enzyme of Rhodothermus obamensis

Background

Glycogen and starch branching enzymes catalyze the formation of α(1 → 6) linkages in storage polysaccharides by rearrangement of preexisting α-glucans. This reaction occurs through the cleavage of α(1 → 4) linkage and transfer in α(1 → 6) of the fragment in non-reducing position. These enzymes define major elements that control the structure of both glycogen and starch.

Methods

The kinetic parameters of the branching enzyme of Rhodothermus obamensis (RoBE) were established after in vitro incubation with different branched or unbranched α-glucans of controlled structure.

Results

A minimal chain length of ten glucosyl units was required for the donor substrate to be recognized by RoBE that essentially produces branches of DP 3–8. We show that RoBE preferentially creates new branches by intermolecular mechanism. Branched glucans define better substrates for the enzyme leading to the formation of hyper-branched particles of 30–70 nm in diameter (dextrins). Interestingly, RoBE catalyzes an additional α-4-glucanotransferase activity not described so far for a member of the GH13 family.

Conclusions

RoBE is able to transfer α(1 → 4)-linked-glucan in C4 position (instead of C6 position for the branching activity) of a glucan to create new α(1 → 4) linkages yielding to the elongation of linear chains subsequently used for further branching. This result is a novel case for the thin border that exists between enzymes of the GH13 family.

General significance

This work reveals the original catalytic properties of the thermostable branching enzyme of R. obamensis. It defines new approach to produce highly branched α-glucan particles in vitro.     

The Role of the Central Globular Domain of Histone H5 in Chromatin Structure

Abstract

Histone H5 contains three tryosines in the central, a polar region of the molecule. All three tryosines can be spin labeled at low ionic strength. When the central globular domain is folded at high ionic strength, only one tyrosine becomes accessible to the imidazole spin label. Spin labeling the buried tyrosines prevents the folding of the globular structure, which, in turn, affects the proper binding of the H5 molecule to stripped chromatin. Chromatin complexes reconstituted from such an extensively modified H5 molecule show a weaker protection of the 168 base pair chromatosome during nuclease digestion. However, when only the surface tyrosine of the H5 molecule is labeled, such a molecule can still bind correctly to stripped chromatin, yielding a complex very similar to that of native chromatin. Our data supports the idea that not just the presence of the linker histone H5, but the presence of an intact H5 molecule with a folded, globular central domain is essential in the recognition of its specific binding sites on the nucleosomes. Our data also show that during the chromatin condensation process, the tumbling environment of the spin label attached to the surface tyrosine in the H5 molecule is not greatly hindered but remains partially mobile. This suggests that either the labeled domain of the H5 molecule is not directly involved in the condensation process or the formation of the higher-order chromatin structure does not result in a more viscous or tighter environment around the spin label. The folded globular domain of H5 molecule serves in stabilizing the nucleosome structure, as well as the higherorder chromatin structure.     

Artificial feeding of Varroa destructor through a chitosan membrane: a tool for studying the host-microparasite relationship

Rearing pests or parasites of very small size in the absence of their living host is a challenge for behavioural, physiological and pathological studies. For feeding Varroa destructor, an ectoparasitic mite of Apis mellifera, a confinement space with a membrane separating the nutritive solution and the space was designed. The mite measures less than 2 mm and bears a perforating apparatus with a length of 15 μm. The membrane, an essential element of the chamber, has a thickness of 0.1 μm, and is made of chitosan. It closes one face of the individual confinement chamber and allows piercing and the ingestion of the nutritive solution. Factors inducing feeding can be applied on the inner walls or on the membrane. In the particular case of Varroa, the highest percentages of feeding mites are obtained by addition of host haemolymph to the nutritive solution, suggesting the kairomonal role of haemolymph in addition to its nutritional one. The membrane concept can be easily applied to several mites or other micro-pests.     

Inhibition of DEPDC1A,a Bad Prognostic Marker in Multiple Myeloma,Delays Growth and Induces Mature Plasma Cell Markers in Malignant Plasma Cells

High throughput DNA microarray has made it possible to outline genes whose expression in malignant plasma cells is associated with short overall survival of patients with Multiple Myeloma (MM). A further step is to elucidate the mechanisms encoded by these genes yielding to drug resistance and/or patients’ short survival. We focus here on the biological role of the DEP (for Disheveled, EGL-10, Pleckstrin) domain contained protein 1A (DEPDC1A), a poorly known protein encoded by DEPDC1A gene, whose high expression in malignant plasma cells is associated with short survival of patients. Using conditional lentiviral vector delivery of DEPDC1A shRNA, we report that DEPDC1A knockdown delayed the growth of human myeloma cell lines (HMCLs), with a block in G2 phase of the cell cycle, p53 phosphorylation and stabilization, and p21^Cip1 accumulation. DEPDC1A knockdown also resulted in increased expression of mature plasma cell markers, including CXCR4, IL6-R and CD38. Thus DEPDC1A could contribute to the plasmablast features of MMCs found in some patients with adverse prognosis, blocking the differentiation of malignant plasma cells and promoting cell cycle.