Attenuation of leakiness in doxycycline-inducible expression via incorporation of 3' AU-rich mRNA destabilizing elements

Tetracycline-regulated expression systems have been widely used for inducible protein expression in cultured mammalian cells. With these systems, however, leakiness in expression of the target gene in the absence of the inducing agent is a frequent problem. Here we describe a novel approach to overcome this problem that involves the incorporation of AU-rich mRNA destabilizing elements (AREs) into the 3' untranslated regions of the tetracycline-inducible constructs. Using the inducible expression of sphingosine kinase 1 and 2 in HEK293 cells as model systems, we found this ARE approach to be remarkably successful in ablating expression of these proteins in the absence of doxycycline through decreasing stability of their mRNAs. We show that this undemanding and flexible process results in a substantial decrease in the leakiness of the tetracycline-inducible expression system while maintaining a high level of target protein expression following induction.     

Islet1 cardiovascular progenitors: a single source for heart lineages?

The creation of regenerative stem cell therapies for heart disease requires that we understand the molecular mechanisms that govern the fates and differentiation of the diverse muscle and non-muscle cell lineages of the heart. Recently, different cardiac cell types have been reported to arise from a common, multipotent Islet1 (Isl1)-positive progenitor, suggesting that a clonal model of heart lineage diversification might occur that is analogous to hematopoiesis. The ability to isolate, renew and differentiate Isl1(+) precursors from postnatal and embryonic hearts and from embryonic stem cells provides a powerful cell-based system for characterizing the signaling pathways that control cardiovascular progenitor formation, renewal, lineage specification and conversion to specific differentiated progeny.     

Comparison of Genotypes Between Environmental and Clinical Isolates of Cryptococcus neoformans var. grubii Based on Microsatellite Patterns

We applied multilocus microsatellite typing (MLMT) method to investigate the genetic relation between Cryptococcus neoformans var. grubii clinical and environmental isolates in S?o Paulo, Brazil. This MLMT method includes three functional gene sequences of C. neoformans var. grubii, which are dispersed on three chromosomes. In all, 89 strains (36 clinical and 53 environmental isolates) were analyzed. Of 36 clinical strains, 20 belonged to a major type of MLMT-13 (55.6%). They were mainly isolated from clinical specimens. About 52.8% of strains from the environment belong to a major type of MLMT-36, which are indigenous to environments and which were not isolated from clinical samples. Thus, we recognized two genotypes that distinguish majority of clinical and environmental strains. No differences were found in antifungal susceptibility and capsule size between major environmental and clinical MLMT types.     

Differential expression of the putative Kex2 processed and secreted aspartic proteinase gene family of Cryphonectria parasitica

Kex2-silenced strains of Cryphonectria parasitica, the ascomycete causal agent of chestnut blight, show a significant reduction in virulence, reduced sexual and asexual sporulation and reductions in mating and fertility. Due to this and the known involvement of Kex2 in the processing of important proproteins in other systems, we searched the whole C. parasitica genome for putative Kex2 substrates. Out of 1299 open reading frames (ORFs) predicted to be secreted, 222 ORFs were identified as potential Kex2 substrates by this screen. Within the putative substrates we identified cell wall modifying proteins, putative proteinases, lipases, esterases, and oxidoreductases. This in silico screen also uncovered a family of nine secreted aspartic proteinases (SAPs) of C. parasitica. Northern blot analyses of this gene family showed differential expression when exposed to chestnut wood and Cryphonectria hypovirus 1 (CHV1). Due to the reduction in fungal virulence known to be caused upon hypoviral infection of C. parasitica, the differential gene expression observed, and the known involvement of SAPs in virulence in other systems, we conducted deletion analyses of four of these proteinases, representing different expression patterns. Deletion of each of the four SAPs did not affect growth rates, sporulation or virulence, suggesting that none of the considered SAPs is essential for the full development or virulence of C. parasitica under the conditions tested.     

Conformational dimorphism of self-peptides and molecular mimicry in a disease-associated HLA-B27 subtype

An interesting property of certain peptides presented by major histocompatibility complex (MHC) molecules is their acquisition of a dual binding mode within the peptide binding groove. Using x-ray crystallography at 1.4 A resolution, we show here that the glucagon receptor-derived self-peptide pGR ((412)RRRWHRWRL(420)) is presented by the disease-associated human MHC class I subtype HLA-B*2705 in a dual conformation as well, with the middle of the peptide bent toward the floor of the peptide binding groove of the molecule in both binding modes. The conformations of pGR are compared here with those of another self-peptide (pVIPR, RRKWRRWHL) that is also displayed in two binding modes by HLA-B*2705 antigens and with that of the viral peptide pLMP2 (RRRWRRLTV). Conserved structural features suggest that the N-terminal halves of the peptides are crucial in allowing cytotoxic T lymphocyte (CTL) cross-reactivity. In addition, an analysis of T cell receptors (TCRs) from pGR- or pVIPR-directed, HLA-B27-restricted CTL clones demonstrates that TCR from distinct clones but with comparable reactivity may share CDR3alpha but not CDR3beta regions. Therefore, the cross-reactivity of these CTLs depends on TCR-CDR3alpha, is modulated by TCR-CDR3beta sequences, and is ultimately a consequence of the conformational dimorphism that characterizes binding of the self-peptides to HLA-B*2705. These results lend support to the concept that conformational dimorphisms of MHC class I-bound peptides might be connected with the occurrence of self-reactive CTL.     

Characterization of articular chondrocytes isolated from 211 osteoarthritic patients

We analyzed specific features of chondrocytes as cellular yield, cell doubling rates and the dependence between these parameters and patient-related data in a set of 211 osteoarthritic (OA) patients undergoing total joint replacement. For each patient the data available were joint type, age and gender. Knee samples chosen randomly among all biopsies were graded according to ICRS score. Patients’ age ranged between 30 and 90 years with a mean age of 66 ± 9.7 years. Patients were divided into age classes and statistically significant differences in proliferation rate at passage 1 were found between chondrocytes derived from young and old donors, with the last ones characterized by a lower proliferation rate. A similar trend was observed for proliferation rate at passage 2. For all the samples, cellular yields ranged between 0.1 and 5.5 million cells/g of tissue. No significant correlation was observed between the level of cartilage degeneration (ICRS score) and cellular yield and proliferation rates. However, in samples with a high degree of cartilage degeneration (ICRS score 4) the cellular yield was lower compared to the other three groups (ICRS scores 1–3). In this study we performed a systematic characterization of basic parameters of chondrocytes originating from a wide group of OA patients. Considering the use of autologous chondrocytes in chondral treatments, the characterization of cell basic features may represent an important step to determine the quality of the cell source which is a major determinant in the outcome of cell-based therapies.     

Two-Photon Bidirectional Control and Imaging of Neuronal Excitability with High Spatial Resolution In Vivo

1. Download high-res image (271KB)
2. Download full-size image