The risk of bovine spongiform encephalopathy propagation was drastically reduced after the European Union (EU) Health Authorities adopted restrictions involving a ban on animal-derived proteins in ...the diet of farm animals. Currently, the EU's officially recommended method for controlling meat and bone meal (MBM) in animal feed is the microscopic method, which involves the identification of bone fragments on the basis of their morphological characteristics. Recently, we demonstrated that a polymerase chain reaction (PCR)-based assay can be used for the detection of taxon-specific DNA in MBM and animal feeds. To ensure the safe rendering of animal by-products, the EU Council requires that this material be treated at 133 degrees C at 300 kPa for 20 min. Here we investigate the relationship between DNA degradation, PCR amplification, and MBM heat treatment. With a competitive PCR-based approach, we compare the amplification efficiency of bovine mitochondrial DNA target sequences of different lengths in several heat-treated MBM samples. For our method, a synthetic competitive DNA is used as an internal control for both DNA extraction and PCR reaction. A correlation between an increase in treatment temperature and a reduction in the size of the target sequences suitable for amplification was observed, suggesting progressive DNA fragmentation due to the temperature. We show that short amplicons (147 bp) can be used to detect the presence of bovine mtDNA in MBM samples treated according to the current European regulations. The use of such a competitive approach to compare amplification efficiency levels of targets of different lengths might represent a useful tool for the determination of both the amount of MBM in animal feeds and its proper heat treatment.
The mechanism by which the mitochondrial large rRNA is involved in the restoration of the pole cell-forming ability in Drosophila embryos is still unknown. We identified a 15-ribonucleotide sequence ...which is conserved from the protobacterium Wolbachia to the higher eukaryotes in domain V of the mitochondrial large rRNA. This short sequence is sufficient to restore pole cell determination in UV-irradiated Drosophila embryos. Here, we provide evidence that the conserved 15-base sequence is sufficient to restore luciferase activity in vitro. Moreover, we show that the internal GAGA sequence is involved in protein binding and that mutations in this tetranucleotide affect the sequence’s ability to restore luciferase activity. The obtained results lead us to propose that mtlrRNA may be involved either in damaged protein reactivation or in protein biosynthesis during pole cell determination.
Craniosynostoses are a heterogeneous group of disorders characterized by premature fusion of cranial sutures. Mutations in fibroblast growth factor receptors (FGFRs) have been associated with a ...number of such conditions. Nevertheless, the cellular mechanism(s) involved remain unknown. We analyzed cell proliferation and differentiation in osteoblasts obtained from patients with three genetically and clinically distinct craniosynostoses: Pfeiffer syndrome carrying the FGFR2 C342R substitution, Apert syndrome with FGFR2 P253R change, and a nonsyndromic craniosynostosis without FGFR canonic mutations, as compared with control osteoblasts. Osteoblasts from craniosynostotic patients exhibited a lower proliferation rate than control osteoblasts. P253R and nonsyndromic craniosynostosis osteoblasts showed a marked differentiated phenotype, characterized by high alkaline phosphatase activity, increased mineralization and expression of noncollagenous matrix proteins, associated with high expression and activation of protein kinase Cα and protein kinase Cε isoenzymes. By contrast, the low proliferation rate of C342R osteoblasts was not associated with a differentiated phenotype. Although they showed higher alkaline phosphatase activity than control, C342R osteoblasts failed to mineralize and expressed low levels of osteopontin and osteonectin and high protein kinase Cζ levels. Stimulation of proliferation and inhibition of differentiation were observed in all cultures on FGF2 treatment. Our results suggest that an anticipated proliferative/differentiative switch, associated with alterations of the FGFR transduction pathways, could be the causative common feature in craniosynostosis and that mutations in distinct FGFR2 domains are associated with an
in vitro heterogeneous differentiative phenotype.
A ban on ruminant-derived proteins in ruminant feeds has been introduced as a preventive measure to avoid the spread of bovine spongiform encephalopathy (BSE), as well as to minimize any potential ...risk of BSE transmission from bovines to humans. In the absence of commercially available efficient methods for identification of bovine-derived proteins in animal feeds, we developed a rapid and sensitive polymerase chain reaction (PCR)-based assay which allows detection and identification of a bovine-specific mitochondrial DNA sequence from feedstuffs. The amplified product encodes for the whole ATPase subunit 8 and amino-terminal portion of the ATPase subunit 6 proteins, which are known to exhibit a relatively low degree of conservation among vertebrates. The specific amplification of such a bovine mitochondrial sequence from reference feedstuff samples was demonstrated by means of both direct sequencing and single-strand conformational analysis of the PCR product. Specificity was also confirmed by the absence of detectable homologous PCR product when using reference feedstuff samples lacking bovine-derived meat and bonemeals, or genomic DNA samples from vertebrates whose offals are commonly included in animal feeds. This method allows detection of the presence of bovine mitochondrial DNA in feedstuffs containing less than 0.125% of bovine-derived meat and bonemeals. Furthermore, it does not appear to be considerably affected by prolonged heat treatment. DpnII and SspI restriction endonuclease digestions of the unpurified PCR product may be used routinely to confirm the bovine origin of the amplified sequence. Since this method is specific, rapid, and sensitive, it could be successfully utilized as a routine control assay to evaluate the presence of bovine-derived meat and bonemeals in ruminant feeds
To analyze the mechanism of Tat-mediated HIV pathogenicity, we produced a Drosophila melanogaster strain transgenic for HIV-tat gene and induced the expression of the protein during Drosophila ...development. By in vitro and in vivo experiments, we demonstrated that Tat specifically binds to tubulin via the MAP-binding domain of tubulin, and that this interaction delays the polymerization of tubulin and induces a premature stop to microtubule-dependent cytoplasmic streaming. The delay in the polymerization of microtubules, the tracks for the transport of the axes determinants, alters the positioning of the dorso-ventral axis as shown by the mislocalization of Gurken and Kinesin in oocyte of Drosophila after Tat induction. These results validate the use of Drosophila as a tool to study the molecular mechanism of viral gene products and suggest that Tat-tubulin interaction is responsible for neurodegenerative diseases associated with AIDS.
CuInS2 quantum dots have emerged in the last years as non-toxic alternative to traditional Pb and Cd based quantum dots, especially for biological applications. In this work, the hydrothermal ...synthesis of alloyed Cu-In-Zn-S quantum dots (CIZS) doped with manganese(II) is explored, with different metal ratios (Mn-CIZSy). The doped quantum dots show the sensitized emission of Mn2+ (approximately ms lifetime), together with the emission of the CIZS structure (approximately µs lifetime). The relative contribution of Mn2+ emission is highly dependent on the composition of the CIZS hosting structure (In:Cu ratio). In addition to that, it is shown that Mn2+ sensitization requires a threshold energy, which suggests the involvement of an intermediate state in the sensitization mechanism. The long-lived emission intensity decay of Mn2+ shows a stable and reversible temperature response in physiological conditions (25-45 °C, pH = 7.4). Mn-CIZSy quantum dots are thus interesting candidates as biological luminescent temperature probe thanks to their easy synthesis, high colloidal stability, insensitivity to dioxygen quenching and quantitative time-gated detection.CuInS2 quantum dots have emerged in the last years as non-toxic alternative to traditional Pb and Cd based quantum dots, especially for biological applications. In this work, the hydrothermal synthesis of alloyed Cu-In-Zn-S quantum dots (CIZS) doped with manganese(II) is explored, with different metal ratios (Mn-CIZSy). The doped quantum dots show the sensitized emission of Mn2+ (approximately ms lifetime), together with the emission of the CIZS structure (approximately µs lifetime). The relative contribution of Mn2+ emission is highly dependent on the composition of the CIZS hosting structure (In:Cu ratio). In addition to that, it is shown that Mn2+ sensitization requires a threshold energy, which suggests the involvement of an intermediate state in the sensitization mechanism. The long-lived emission intensity decay of Mn2+ shows a stable and reversible temperature response in physiological conditions (25-45 °C, pH = 7.4). Mn-CIZSy quantum dots are thus interesting candidates as biological luminescent temperature probe thanks to their easy synthesis, high colloidal stability, insensitivity to dioxygen quenching and quantitative time-gated detection.
Inside the cell, the HIV Tat protein is mainly found in the nucleus and nucleolus. The nucleolus, the site of ribosome biogenesis, is a highly organized, non-membrane-bound sub-compartment where ...proteins with a high affinity for nucleolar components are found. While it is well known that Tat accumulates in the nucleolus via a specific nucleolar targeting sequence, its function in this compartment it still unknown.
To clarify the significance of the Tat nucleolar localization, we induced the expression of the protein during oogenesis in Drosophila melanogaster strain transgenic for HIV-tat gene. Here we show that Tat localizes in the nucleoli of Drosophila oocyte nurse cells, where it specifically co-localizes with fibrillarin. Tat expression is accompanied by a significant decrease of cytoplasmic ribosomes, which is apparently related to an impairment of ribosomal rRNA precursor processing. Such an event is accounted for by the interaction of Tat with fibrillarin and U3 snoRNA, which are both required for pre-rRNA maturation.
Our data contribute to understanding the function of Tat in the nucleolus, where ribosomal RNA synthesis and cell cycle control take place. The impairment of nucleolar pre-rRNA maturation through the interaction of Tat with fibrillarin-U3snoRNA complex suggests a process by which the virus modulates host response, thus contributing to apoptosis and protein shut-off in HIV-uninfected cells.
The four members of the fibroblast growth factor receptor (FGFR) family are cell-surface membrane-spanning tyrosine kinase receptors involved in a wide spectrum of biologic processes. Much evidence ...also indicates that mutations in FGFR genes result in several craniosynostotic disorders and chondrodysplasias, and that changes in qualitative and quantitative FGFR expression profiles are implicated in tumor induction or progression. Here, we describe a precise and reliable competitive PCR-based assay to evaluate human FGFR1-4 gene expression. A single multispecific synthetic competitive template was designed to amplify FGFR1-4 homologous stretches and constructed to contain FGFR1/FGFR2/FGFR3/FGFR4/GAPDH tandemly arranged forward and reverse primers that allow competition for cDNA-specific primer annealing. The housekeeping GAPDH transcript was utilized as a reference for comparing the expression profiles of different RNA pools. The assay herein described allows the comparison of relative FGFR expression levels, both within a single RNA pool and among multiple RNA pool samples. The major advantages of such a PCR-based approach are its ability to obtain unbiased FGFR mRNA expression patterns and to detect transcripts present in low copy number. Qualitative and semiquantitative analyses of the FGFR1-4 transcript repertoire in mesenchymal- and epithelial-derived primary cell cultures and cell lines demonstrated the utility of such a method to investigate the FGFR1-4 functional role in FGF signal transduction.
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