Mutations in IMPG2, Encoding Interphotoreceptor Matrix Proteoglycan 2, Cause Autosomal-Recessive Retinitis Pigmentosa

Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal diseases caused by progressive degeneration of the photoreceptor cells. Using autozygosity mapping, we identified two families, each with three affected siblings sharing large overlapping homozygous regions that harbored the IMPG2 gene on chromosome 3. Sequence analysis of IMPG2 in the two index cases revealed homozygous mutations cosegregating with the disease in the respective families: three affected siblings of Iraqi Jewish ancestry displayed a nonsense mutation, and a Dutch family displayed a 1.8 kb genomic deletion that removes exon 9 and results in the absence of seven amino acids in a conserved SEA domain of the IMPG2 protein. Transient transfection of COS-1 cells showed that a construct expressing the wild-type SEA domain is properly targeted to the plasma membrane, whereas the mutant lacking the seven amino acids appears to be retained in the endoplasmic reticulum. Mutation analysis in ten additional index cases that were of Dutch, Israeli, Italian, and Pakistani origin and had homozygous regions encompassing IMPG2 revealed five additional mutations; four nonsense mutations and one missense mutation affecting a highly conserved phenylalanine residue. Most patients with IMPG2 mutations showed an early-onset form of RP with progressive visual-field loss and deterioration of visual acuity. The patient with the missense mutation, however, was diagnosed with maculopathy. The IMPG2 gene encodes the interphotoreceptor matrix proteoglycan IMPG2, which is a constituent of the interphotoreceptor matrix. Our data therefore show that mutations in a structural component of the interphotoreceptor matrix can cause arRP.     

Novel flow cytometric approach for the detection of adipocyte subpopulations during adipogenesis

The ability of mesenchymal stromal cells (MSCs) to differentiate into adipocytes provides a cellular model of human origin to study adipogenesis in vitro. One of the major challenges in studying adipogenesis is the lack of tools to identify and monitor the differentiation of various subpopulations within the heterogeneous pool of MSCs. Cluster of differentiation (CD)36 plays an important role in the formation of intracellular lipid droplets, a key characteristic of adipocyte differentiation/maturation. The objective of this study was to develop a reproducible quantitative method to study adipocyte differentiation by comparing two lipophilic dyes [Nile Red (NR) and Bodipy 493/503] in combination with CD36 surface marker staining. We identified a subpopulation of adipose-derived stromal cells that express CD36 at intermediate/high levels and show that combining CD36 cell surface staining with neutral lipid-specific staining allows us to monitor differentiation of adipose-derived stromal cells that express CD36^{intermediate/high} during adipocyte differentiation in vitro. The gradual increase of CD36^{intermediate/high/}NR^positive cells during the 21 day adipogenesis induction period correlated with upregulation of adipogenesis-associated gene expression.     

Distinct eRF3 requirements suggest alternate eRF1 conformations mediate peptide release during eukaryotic translation termination

Organisms that use the standard genetic code recognize UAA, UAG, and UGA as stop codons, whereas variant code species frequently alter this pattern of stop codon recognition. We previously demonstrated that a hybrid eRF1 carrying the Euplotes octocarinatus domain 1 fused to Saccharomyces cerevisiae domains 2 and 3 (Eo/Sc eRF1) recognized UAA and UAG, but not UGA, as stop codons. In the current study, we identified mutations in Eo/Sc eRF1 that restore UGA recognition and define distinct roles for the TASNIKS and YxCxxxF motifs in eRF1 function. Mutations in or near the YxCxxxF motif support the cavity model for stop codon recognition by eRF1. Mutations in the TASNIKS motif eliminated the eRF3 requirement for peptide release at UAA and UAG codons, but not UGA codons. These results suggest that the TASNIKS motif and eRF3 function together to trigger eRF1 conformational changes that couple stop codon recognition and peptide release during eukaryotic translation termination.     

Bimodal effect of humic acids on the LPS-induced TNF-α release from differentiated U937 cells

Humic substances (HS) have been reported to possess anti-inflammatory as well as pro-inflammatory properties. The anti-inflammatory activity was demonstrated in the rat paw edema model and we found a preliminary explanation in the 5-lipoxygenase inhibitory effect of humic acids (HA). The pro-inflammatory activity is reflected by the production and release of pro-inflammatory cytokines in HA-treated neutrophilic granulocytes. With regard to the potential use of HA as antiviral and UV-protective agents it appears advisable to investigate the role of HS in the inflammation process in more detail. Hence we tested four different HS preparations – two naturally occurring HA from the Altteich peatland in Germany, one fulvic acid (FA) preparation from a Finnish spruce forest and a synthetic HA-like polymer (caffeic acid oxidation product, KOP) for their influence on the lipopolysaccharide (LPS)-induced TNF-α release in human U937 cells. In addition, the cytotoxicity of HS was determined.The results demonstrate a concentration-dependent bimodal effect of HA on the TNF-α release of differentiated LPS-stimulated U937 cells for both the natural black peat HA from the Altteich peatland and the HA-like polymer KOP. Low HA concentrations (10–80 μg/ml) enhanced the TNF-α release by up to threefold (pro-inflammatory activity), while HA concentrations >100 μg/ml reduced it about 10-fold (anti-inflammatory activity). FA failed to enhance TNF-α release, but reduced it at higher concentrations (>200 μg/ml) by the half. Brown water HA did not exert any significant effect on TNF-α release. No HS-stimulated TNF-α release was also observed in the absence of exogenously supplied LPS. This means that HS, unlike endotoxin, are no inflammation-causing agents for LPS-untreated cells. Differences in the effect of individual HS on TNF-α release are discussed in connection with the polyanionic character of HS, their molecular mass distribution and the hitherto imperfectly known chemical structure.     

Influence of materials and precoating on initial anaerobic biofilm development

Summary This study investigated the possibility of reducing anaerobic reactor start-up times through the use of various support surfaces or through the use of biological precoating (denitrifying biofilm) and chemical precoating (polymer precoating) as means of enhancing anaerobic biofilm development. Results from the support media variation experiment indicated significant differences among the materials. Support media precoating with denitrifying bacterial biofilms and the various polymers tested did not appear to enhance the rate of initial anaerobic biofilm accumulation.     

Hemodynamic physiology and thermoregulation in liposuction

Little is known about the physiology of large-volume liposuction. Patients are exposed to prolonged procedures, general anesthesia, fluid shifts, and infusion of high doses of epinephrine and lidocaine. Consequently, the authors examined the thermoregulatory and cardiovascular responses to liposuction by assessing multiple physiologic factors. The aims of their study were to serially determine hemodynamic parameters perioperatively, to quantify perioperative and postoperative plasma epinephrine levels, and to chronologically document fluctuations in core body temperature. Five female volunteers with American Society of Anesthesiologists' physical status I and II underwent moderate- to large-volume liposuction. Heart rate, blood pressure, mean pulmonary arterial pressure, cardiac index, and central venous pressure were monitored. Serum epinephrine levels and core body temperature were assessed perioperatively. The hemodynamic responses to liposuction were characterized by an increase in cardiac index (57 percent), heart rate (47 percent), and mean pulmonary arterial pressure (44 percent) (p < 0.05). Central venous pressure was not significantly altered. Maximum epinephrine levels were observed 5 to 6 hours after induction. Significant correlations between cardiac index and epinephrine concentrations were shown intraoperatively (r = 0.75). All patients developed intraoperative low body temperatures (mean 35.5 degrees C). An overall enhanced cardiac function was observed in patients subsequent to large-volume liposuction. The etiology of the altered cardiac parameters was multifactorial but may have been attributable in part to the administration of epinephrine, which counters the effects of general anesthesia and operative hypothermia. Additional explanations for raised cardiac output may be hemodilution or emergence from general anesthesia. Elevated mean pulmonary arterial pressure may be a result of subclinical fat embolism demonstrated in previous porcine studies, although fat was not observed in urine. The unchanged central venous pressure levels indicate that young healthy patients with compliant right ventricles can accommodate the fluid loads of large-volume liposuction. Overall hemodynamic parameters remained within safe limits. Within these surgical parameters, patients should be clinically screened for cardiovascular and blood pressure disorders before liposuction is undertaken, and preventative measures should be taken to limit intraoperative hypothermia.     

CC2D2A is mutated in Joubert syndrome and interacts with the ciliopathy-associated basal body protein CEP290

Joubert syndrome and related disorders (JSRD) are primarily autosomal-recessive conditions characterized by hypotonia, ataxia, abnormal eye movements, and intellectual disability with a distinctive mid-hindbrain malformation. Variable features include retinal dystrophy, cystic kidney disease, and liver fibrosis. JSRD are included in the rapidly expanding group of disorders called ciliopathies, because all six gene products implicated in JSRD (NPHP1, AHI1, CEP290, RPGRIP1L, TMEM67, and ARL13B) function in the primary cilium/basal body organelle. By using homozygosity mapping in consanguineous families, we identify loss-of-function mutations in CC2D2A in JSRD patients with and without retinal, kidney, and liver disease. CC2D2A is expressed in all fetal and adult tissues tested. In ciliated cells, we observe localization of recombinant CC2D2A at the basal body and colocalization with CEP290, whose cognate gene is mutated in multiple hereditary ciliopathies. In addition, the proteins can physically interact in vitro, as shown by yeast two-hybrid and GST pull-down experiments. A nonsense mutation in the zebrafish CC2D2A ortholog (sentinel) results in pronephric cysts, a hallmark of ciliary dysfunction analogous to human cystic kidney disease. Knockdown of cep290 function in sentinel fish results in a synergistic pronephric cyst phenotype, revealing a genetic interaction between CC2D2A and CEP290 and implicating CC2D2A in cilium/basal body function. These observations extend the genetic spectrum of JSRD and provide a model system for studying extragenic modifiers in JSRD and other ciliopathies.     

Distinct paths to stop codon reassignment by the variant-code organisms Tetrahymena and Euplotes

The reassignment of stop codons is common among many ciliate species. For example, Tetrahymena species recognize only UGA as a stop codon, while Euplotes species recognize only UAA and UAG as stop codons. Recent studies have shown that domain 1 of the translation termination factor eRF1 mediates stop codon recognition. While it is commonly assumed that changes in domain 1 of ciliate eRF1s are responsible for altered stop codon recognition, this has never been demonstrated in vivo. To carry out such an analysis, we made hybrid proteins that contained eRF1 domain 1 from either Tetrahymena thermophila or Euplotes octocarinatus fused to eRF1 domains 2 and 3 from Saccharomyces cerevisiae. We found that the Tetrahymena hybrid eRF1 efficiently terminated at all three stop codons when expressed in yeast cells, indicating that domain 1 is not the sole determinant of stop codon recognition in Tetrahymena species. In contrast, the Euplotes hybrid facilitated efficient translation termination at UAA and UAG codons but not at the UGA codon. Together, these results indicate that while domain 1 facilitates stop codon recognition, other factors can influence this process. Our findings also indicate that these two ciliate species used distinct approaches to diverge from the universal genetic code.