Expression of rod-derived cone viability factor: dual role of CRX in regulating promoter activity and cell-type specificity

Background

RdCVF and RdCVF2, encoded by the nucleoredoxin-like genes NXNL1 and NXNL2, are trophic factors with therapeutic potential that are involved in cone photoreceptor survival. Studying how their expression is regulated in the retina has implications for understanding both their activity and the mechanisms determining cell-type specificity within the retina.

Methodology/Principal Findings

In order to define and characterize their promoters, a series of luciferase/GFP reporter constructs that contain various fragments of the 5′-upstream region of each gene, both murine and human, were tested in photoreceptor-like and non-photoreceptor cell lines and also in a biologically more relevant mouse retinal explant system. For NXNL1, 5′-deletion analysis identified the human −205/+57 bp and murine −351/+51 bp regions as having promoter activity. Moreover, in the retinal explants these constructs drove expression specifically to photoreceptor cells. For NXNL2, the human −393/+27 bp and murine −195/+70 bp regions were found to be sufficient for promoter activity. However, despite the fact that endogenous NXNL2 expression is photoreceptor-specific within the retina, neither of these DNA sequences nor larger upstream regions demonstrated photoreceptor-specific expression. Further analysis showed that a 79 bp NXNL2 positive regulatory sequence (−393 to 315 bp) combined with a 134 bp inactive minimal NXNL1 promoter fragment (−77 to +57 bp) was able to drive photoreceptor-specific expression, suggesting that the minimal NXNL1 fragment contains latent elements that encode cell-type specificity. Finally, based on bioinformatic analysis that suggested the importance of a CRX binding site within the minimal NXNL1 fragment, we found by mutation analysis that, depending on the context, the CRX site can play a dual role.

Conclusions/Significance

The regulation of the Nucleoredoxin-like genes involves a CRX responsive element that can act as both as a positive regulator of promoter activity and as a modulator of cell-type specificity.     

Contribution of NZB autoimmunity 2 to Y-linked autoimmune acceleration-induced monocytosis in association with murine systemic lupus

The accelerated development of systemic lupus erythematosus (SLE) in BXSB male mice is associated with the presence of the Y-linked autoimmune acceleration (Yaa) mutation, which induces an age-dependent monocytosis. Using a cohort of C57BL/6 (B6) x (NZB x B6)F1 backcross male mice bearing the Yaa mutation, we defined the pathogenic role and genetic basis for Yaa-associated monocytosis. We observed a remarkable correlation of monocytosis with autoantibody production and subsequent development of lethal lupus nephritis, indicating that monocytosis is an additional useful indicator for severe SLE. In addition, we identified an NZB-derived locus on chromosome 1 predisposing to the development of monocytosis, which peaked at Fcgr2b encoding FcgammaRIIB and directly overlapped with the previously identified NZB autoimmunity 2 (Nba2) locus. The contribution of Nba2 to monocytosis was confirmed by the analysis of Yaa-bearing B6 mice congenic for the NZB-Nba2 locus. Finally, we observed a very low-level expression of FcgammaRIIB on macrophages bearing the NZB-type Fcgr2b allele, compared with those bearing the B6-type allele, and the development of monocytosis in FcgammaRIIB haploinsufficient B6 mice carrying the Yaa mutation. These data suggest that the Nba2 locus may play a supplementary role in the pathogenesis of SLE by promoting the development of monocytosis and the activation of effector cells bearing stimulatory FcgammaR, in addition to its implication in the dysregulated activation of autoreactive B cells.     

Energetics of the growth of Methanococcus thermolithotrophicus

Methanococcus thermolithotrophicus was grown in a mineral salts medium at 65° C in a fermenter gassed with H₂ and CO₂, which were the sole carbon and energy sources. Evolution of growth parameters during batch culture experiments showed the existence of an uncoupling phenomenon. The growth was then studied using a continuous technique and steady states for various gas flow rates were obtained. Y _{CH 4}and the maintenance coefficient varied with the gas input. The maximum Y _{CH 4} determined for Methanococcus thermolithotrophicus was 3.33 g·mol^-1 CH₄. An excess of energy and carbon sources induced uncoupling of growth.     

Spectrofluorimetric methods for the measurement of the so called ”Microviscosity“ of lipidic structures

The cellular interactions involving the membrane depend on its physico-chemical nature and on the topographical distribution of the membrane receptors. At present, the role of the lipidic regions is not well defined; however, it is known that the fluidity or « microviscosityof the lipidic components controls important processes in cellular biology.Different spectrofluorimetric methods, continuous or time resolved, susceptible to the cohesion of lipidic regions have been developed:

1. The anisotropy of fluorescence where the rotation of probes is studied (linked to the coefficient of diffusional rotation).

2. The inhibition of fluorescence where the kinetics of the reaction is practically diffusion controlled.

3. The formation of emissive intramolecular complexes where the internal rotations of interchromophoric bonds are studied.

After the development of kinetic models, the methods have been tested with synthetic and natural organized assemblies.The values of « microviscosityobtained with these methods may be different because the environments of probes are different. Therefore, the concept of « microviscosity , applied to biological membranes is limited.     

Electrophoretic Transfer from Polyacrylamide Gel to Nitrocellulose Sheets, a New Method To Characterize Multilocus Enzyme Genotypes of Klebsiella Strains

A new method for multilocus enzyme electrophoresis, based on electrophoretic transfers to nitrocellulose after polyacrylamide-agarose gel electrophoresis was explored. Electrophoretic separation was performed on 1-mm-thick slab gels with 6-μl samples of bacterial extracts and was followed by serial 5-min consecutive transfers. The transferability of 19 metabolic enzymes of Klebsiella strains was studied and allowed the simultaneous examination of one enzyme in the separation gel and at least five enzymes on nitrocellulose sheets. The resolution of enzyme bands was increased on nitrocellulose; thus, well-separated bands were recorded for nucleoside phosphorylase, peptidase, and phosphoglucose isomerase whereas their mobility variants could not be clearly distinguished in the separation gel because of stain diffusion. The study of genetic relationships of 42 strains of Klebsiella pneumoniae and 24 strains of Klebsiella oxytoca demonstrated the reliability of the method, since clustering analysis of electrophoretic types, based on electrophoretic polymorphism of 10 metabolic enzymes, showed two main clusters well correlated with the two species. The 57 electrophoretic types described confirm the usefulness of the method for the study of genetic relationships between closely related strains.     

Monoclonal Anti-Conjugated Acetylcholine Antibody and Immunohistochemical Applications in Rat Nervous System

Acetylcholine (ACh) conjugates were injected into AKR and DBA mice over a period of 10 weeks. The polyclonal antisera were tested at various immunization times for affinity and specificity using an enzyme-linked immunosorbent assay (ELISA). The most immunoreactive compound was found to be choline-glutaryl-bovine serum albumin (or conjugated ACh). The AKR and DBA mice yielding the highest apparent affinity were killed, and the spleen cells were fused with X63 or SP2/O/Ag mouse myeloma cells. Supernatants of confluent cultures were tested for the presence of anti-conjugated ACh antibodies using the same ELISA method. The best results were obtained with the hybridomas from AKR spleen cells and X63 mouse myeloma cells. Monoclonal antibody affinity and specificity were then evaluated by a radioimmunological procedure using iodinated monoclonal anti-conjugated ACh antibody. From competition experiments, the most immunoreactive compound was choline-glutaryl-protein. The other related compounds were recognized either poorly or not at all. The high affinity and specificity of our monoclonal antibody enabled us to visualize ACh molecules on fixed rat brain sections. ACh was fixed with a mixture of nitrobenzyl alcohol and glutaraldehyde. Many ACh-immunoreactive cell bodies and fibers were seen on sections from the basal forebrain and spinal cord. Preadsorption and other immunohistochemical tests demonstrated that the ACh staining was highly specific.     

Plant N-glycan processing enzymes employ different targeting mechanisms for their spatial arrangement along the secretory pathway

The processing of N-linked oligosaccharides in the secretory pathway requires the sequential action of a number of glycosidases and glycosyltransferases. We studied the spatial distribution of several type II membrane-bound enzymes from Glycine max, Arabidopsis thaliana, and Nicotiana tabacum. Glucosidase I (GCSI) localized to the endoplasmic reticulum (ER), alpha-1,2 mannosidase I (ManI) and N-acetylglucosaminyltransferase I (GNTI) both targeted to the ER and Golgi, and beta-1,2 xylosyltransferase localized exclusively to Golgi stacks, corresponding to the order of expected function. ManI deletion constructs revealed that the ManI transmembrane domain (TMD) contains all necessary targeting information. Likewise, GNTI truncations showed that this could apply to other type II enzymes. A green fluorescent protein chimera with ManI TMD, lengthened by duplicating its last seven amino acids, localized exclusively to the Golgi and colocalized with a trans-Golgi marker (ST52-mRFP), suggesting roles for protein-lipid interactions in ManI targeting. However, the TMD lengths of other plant glycosylation enzymes indicate that this mechanism cannot apply to all enzymes in the pathway. In fact, removal of the first 11 amino acids of the GCSI cytoplasmic tail resulted in relocalization from the ER to the Golgi, suggesting a targeting mechanism relying on protein-protein interactions. We conclude that the localization of N-glycan processing enzymes corresponds to an assembly line in the early secretory pathway and depends on both TMD length and signals in the cytoplasmic tail.