K2P18.1 translates T cell receptor signals into thymic regulatory T cell development

It remains largely unclear how thymocytes translate relative differences in T cell receptor (TCR) signal strength into distinct developmental programs that driv...     

RIC-3 Exclusively Enhances the Surface Expression of Human Homomeric 5-Hydroxytryptamine Type 3A (5-HT3A) Receptors Despite Direct Interactions with 5-HT3A, -C, -D, and -E Subunits

Although five 5-hydroxytryptamine type 3 (5-HT3) subunits (A–E) have been cloned, knowledge on the regulation of their assembly is limited. RIC-3 has been identified as a chaperone specific for the pentameric ligand-gated nicotinic acetylcholine and 5-HT₃ receptors. Therefore, we examined the impact of RIC-3 on differently composed 5-HT₃ receptors with the focus on 5-HT3C, -D, and -E subunits. The influence of RIC-3 on these receptor subtypes is supported by the presence of RIC3 mRNA in tissues expressing at least one of the subunits 5-HT3C, -D, and -E. Furthermore, immunocytochemical studies on transfected mammalian cells revealed co-localization in the endoplasmic reticulum and direct interaction of RIC-3 with 5-HT3A, -C, -D, and -E. Functional and pharmacological characterization was performed using HEK293 cells expressing 5-HT3A or 5-HT3A + 5-HT3B (or -C, -D, or -E) in the presence or absence of RIC-3. Ca²⁺ influx analyses revealed that RIC-3 does not influence the 5-HT concentration-response relationship on 5-HT₃A receptors but leads to differential increases of 5-HT-induced maximum response (E_max) on cells expressing different subunits. Increases of E_max were due to analogously enhanced B_max values for binding of the 5-HT₃ receptor antagonist [³H]GR65630. The observed enhanced cell surface expression of the tested 5-HT3 subunit combinations correlated with the increased surface expression of 5-HT3A as determined by flow cytometry. In conclusion, we showed that RIC-3 can interact with 5-HT3A, -C, -D, and -E subunits and predominantly enhances the surface expression of homomeric 5-HT₃A receptors in HEK293 cells. These data implicate a possible role of RIC-3 in determining 5-HT₃ receptor composition in vivo.     

Progression of Parkinson's Disease Pathology Is Reproduced by Intragastric Administration of Rotenone in Mice

In patients with Parkinson''s disease (PD), the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS), the dorsal motor nucleus of the vagus (DMV), the intermediolateral nucleus of the spinal cord and the substantia nigra, providing the basis for the neuropathological staging of the disease. Here we report that intragastrically administered rotenone, a commonly used pesticide that inhibits Complex I of the mitochondrial respiratory chain, is able to reproduce PD pathological staging as found in patients. Our results show that low doses of chronically and intragastrically administered rotenone induce alpha-synuclein accumulation in all the above-mentioned nervous system structures of wild-type mice. Moreover, we also observed inflammation and alpha-synuclein phosphorylation in the ENS and DMV. HPLC analysis showed no rotenone levels in the systemic blood or the central nervous system (detection limit [rotenone]<20 nM) and mitochondrial Complex I measurements showed no systemic Complex I inhibition after 1.5 months of treatment. These alterations are sequential, appearing only in synaptically connected nervous structures, treatment time-dependent and accompanied by inflammatory signs and motor dysfunctions. These results strongly suggest that the local effect of pesticides on the ENS might be sufficient to induce PD-like progression and to reproduce the neuroanatomical and neurochemical features of PD staging. It provides new insight into how environmental factors could trigger PD and suggests a transsynaptic mechanism by which PD might spread throughout the central nervous system.     

The combination of photogrammetry and finite elements for a fine grained functional analysis of anatomical structures

Summary A new method of functional morphological analysis is presented. Combining stereophotogrammetry with the finite element technique, a new approach, permits a three-dimensional numerical stress analysis of arbitrarily shaped bodies to be performed. The stereophotogrammetric method which originated for three-dimensional calculations in the study of surfaces in land surveying is well suited for the determination of the nodal co-ordinates required for the finite element method, an engineering technique developed for behavioural analysis of solids and fluids responding to external forces. This approach was tested in a study of the functional morphology of the bill of an African wading bird, the shoebill Balaeniceps rex. A few findings of that study are given here in order to demonstrate the method. Advantages of the finite element method compared with other techniques for stress analysis of anatomical structures are also discussed. The method presents exciting possibilities for predicting displacement and stress responses more accurately and in much greater detail. The scope of this powerful computerized stress analysis technique is greatly enhanced with the introduction of stereophotogrammetry for determining the three-dimensional co-ordinates of complex anatomical structures. With the finite element method, the properties of the bone structure can be modelled as they occur in the life of the animal. This is not possible with physical models. Furthermore, rare specimens can be analysed non-destructively.     

Identification of protective epitopes on ebola virus glycoprotein at the single amino acid level by using recombinant vesicular stomatitis viruses

Ebola virus causes lethal hemorrhagic fever in humans, but currently there are no effective vaccines or antiviral compounds for this infectious disease. Passive transfer of monoclonal antibodies (MAbs) protects mice from lethal Ebola virus infection (J. A. Wilson, M. Hevey, R. Bakken, S. Guest, M. Bray, A. L. Schmaljohn, and M. K. Hart, Science 287:1664-1666, 2000). However, the epitopes responsible for neutralization have been only partially characterized because some of the MAbs do not recognize the short synthetic peptides used for epitope mapping. To identify the amino acids recognized by neutralizing and protective antibodies, we generated a recombinant vesicular stomatitis virus (VSV) containing the Ebola virus glycoprotein-encoding gene instead of the VSV G protein-encoding gene and used it to select escape variants by growing it in the presence of a MAb (133/3.16 or 226/8.1) that neutralizes the infectivity of the virus. All three variants selected by MAb 133/3.16 contained a single amino acid substitution at amino acid position 549 in the GP2 subunit. By contrast, MAb 226/8.1 selected three different variants containing substitutions at positions 134, 194, and 199 in the GP1 subunit, suggesting that this antibody recognized a conformational epitope. Passive transfer of each of these MAbs completely protected mice from a lethal Ebola virus infection. These data indicate that neutralizing antibody cocktails for passive prophylaxis and therapy of Ebola hemorrhagic fever can reduce the possibility of the emergence of antigenic variants in infected individuals.     

Molecular cloning of the ribosomal RNA genes of the photosynthetic bacterium Rhodopseudomonas capsulata

Summary Chromosomal segments of Rhodopseudomonas capsulata carrying the ribosomal operons and cloned with the cosmid vector pHC79 have been identified by cross hybridization with ³²P-ATP labeled rRNAs. At least seven rRNA operons are present in the R. capsulata chromosome. By R-loop analyses of DNA-RNA hybrids, two distinct loop structures of sizes 1.50 kb and 2.52 kb corresponding to the 16S and 23S RNA molecules, respectively, were detected. Intact 23S RNA molecules can be isolated from R. capsulata ribosomes by sucrose density centrifugation. However, fragmentation of the 23S RNA molecule into a 16S-like molecule was observed during gel electrophoresis. Restriction mapping and hybridization of a 9 kb PstI fragment that contained one copy of the rRNA operon showed the following sequence of the RNA genes in R. capsulata 16S, 23S, and 5S. A spacer region of 0.91 kb was found between the 16S and the 23S RNA genes.     

Purification and PCR-based cDNA cloning of a plastidial n-6 desaturase

A plastidial membrane-bound n-6 desaturase from spinach (Spinacia oleracea) was purified from chloroplast envelope membranes by anion exchange, cation exchange and ferredoxin-affinity chromatography. The molecular mass of the protein was estimated by SDS-PAGE to be 40 kDa. The highest specific activity of the desaturase in the final preparation was 196 nmol/min per mg protein with free oleic acid as the substrate. The N-terminal amino acid sequence of the blotted protein was determined and used for the construction of a degenerated and inosine-containing oligonucleotide primer for PCR experiments with cDNA transcribed from leaf mRNA. A 3-RACE experiment with this primer amplified a single band of 1500 bp that after sequencing showed an open reading frame of 382 amino acids corresponding to a protein of 43 kDa. The 5 end of the cDNA was amplified by a 5-RACE experiment and isolated as a 500 bp fragment. Sequencing of this DNA revealed an additional 65 amino acids at the N-terminus of the native protein that are attributed to a plastidial leader peptide. With appropriate primers derived from these sequences a full-length clone was amplified by PCR and sequenced. Comparison of the plastidial oleate desaturase with the homologous enzyme from cyanobacteria showed about 50% amino acid homology. Comparison with other desaturases revealed three histidine boxes with the general sequence HXXXH that are highly conserved in all membrane-bound desaturases. These boxes might be involved in metal ion complexation required for reduction of oxygen.     

Mutational and structural studies of the diisopropylfluorophosphatase from Loligo vulgaris shed new light on the catalytic mechanism of the enzyme

The active site, the substrate binding site, and the metal binding sites of the diisopropylfluorophosphatase (DFPase) from Loligo vulgaris have been modified by means of site-directed mutagenesis to improve our understanding of the reaction mechanism. Enzymatic characterization of mutants located in the major groove of the substrate binding pocket indicates that large hydrophobic side chains at these positions are favorable for substrate turnover. Moreover, the active site residue His287 proved to be beneficial, but not essential, for DFP hydrolysis. In most cases, hydrophobic side chains at position 287 led to significant catalytic activities although reduced relative to the wild-type enzyme. With respect to the Ca-1 binding site, where catalysis occurs, various mutants indicated that the net charge at this calcium-binding site as well as the relative positions of the charged calcium ligands is crucial for catalytic activity. The importance of the electrostatic potential at the active site was furthermore revealed by various mutations of residues lining the interior of the central water-filled tunnel, which traverses the entire protein structure. In this respect, the structural features of residue His181, which is located at the opposite end of the DFPase tunnel relative to the active site, were characterized extensively. It was concluded that a tunnel-spanning hydrogen bond network, which includes a large number of apparently slow exchanging water molecules, relays any modifications in the electrostatics of the system to the active site, thus affecting the catalytic reactivity of the enzyme.