Martin Radrizzani

Tyrosine Kinase c-Src Constitutes a Bridge between Cystic Fibrosis Transmembrane Regulator Channel Failure and MUC1 Overexpression in Cystic Fibrosis

Cystic fibrosis (CF), a disease caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) chloride channel, is associated in the respiratory system with the accumulation of mucus and impaired lung function. The role of the CFTR channel in the regulation of the intracellular pathways that determine the overexpression of mucin genes is unknown. Using differential display, we have observed the differential expression of several mRNAs that may correspond to putative CFTR-dependent genes. One of these mRNAs was further characterized, and it corresponds to the tyrosine kinase c-Src. Additional results suggest that c-Src is a central element in the pathway connecting the CFTR channel with MUC1 overexpression and that the overexpression of mucins is a primary response to CFTR malfunction in cystic fibrosis, which occurs even in the absence of bacterial infection.

Early effects of insulin-like growth factor-1 in activated human T lymphocytes

This study evaluates the effects of insulin‐like growth factor (IGF)‐1 receptor (IGF‐1R) down‐regulation in stimulated T lymphocytes by investigating the expression of early activation proteins CD69, CD25, and interleukin (IL)‐2. We found that IGF‐1 does not modify CD69 expression but increases transcription and protein synthesis of CD25 and IL‐2. The lowest level of IGF‐1R detected after 15 min of activation suggested that the effects of IGF‐1 occur at the initiation of cell activation. The activation of IGF‐1R was confirmed by IGF‐1R phosphorylation and increased phosphorylation of microtubule‐associated protein kinase. We also detected the alternative IGF‐1 transcripts Ea, with paracrine/autocrine regulation, and Eb, with endocrine regulation, in Jurkat cells and in quiescent T lymphocytes, and we detected IGF‐1 protein in the culture medium after stimulation. These data suggest that the proliferative effects of IGF‐1 on T lymphocytes include both autocrine/paracrine and endocrine processes.

Differential expression of CPD1 during postnatal development in the mouse cerebellum

Several regulated mRNAs were detected by applying differential display to the mouse cerebellum during postnatal development. One cDNA fragment, referred to as CPD1 (GenBank U89345), was characterized and cloned. Northern blots showed maximum mRNA expression at postnatal day seven (P7). The mRNA encodes a protein of 260 amino acids. In situ RT-PCR showed that CPD1 is expressed mainly in granule cells and faintly in Purkinje cells. Polyclonal rabbit antibodies and oligobodies (oligonucleotide-based synthetic antibodies) revealed a protein of 34 kDa in Western blots. Immunohistochemistry showed not only marked nuclear staining but also mild cytoplasmic localization. Granule cells undergoing active division (P4) showed very little expression of CPD1 protein, which increases from P7 to P17. CPD1, affinity-purified using a chemically synthesized oligobody inhibits the activity of protein phosphatase PP2A but not protein phosphatase PP1. Differentiated PC12 cells also showed nuclear and cytoplasmic localization. Interestingly, maximal cytoplasmic CPD1/PP2A colocalization was observed near cell membrane regions that are far from growing neurites, and on growing cones. These results suggest that CPD1 might have an important role in cerebellar development.

Aptamers: current challenges and future prospects.

ABSTRACT Introduction: Aptamers are oligonucleotide molecules raised in vitro from large combinatorial libraries of nucleic acids and developed to bind to targets with high affinity and specificity. Whereas novel target molecules are proposed for therapeutic intervention and diagnostic, aptamer technology has a great potential to become a source of lead compounds. Areas covered: In this review, the authors address the current status of the technology and highlight the recent progress in aptamer-based technologies. They also discuss the current major technical limitations of aptamer technology and propose original solutions based on existing technologies that could result in a solid aptamer-discovery platform. Expert opinion: Whereas aptamers have shown to bind to targets with similar affinities and specificities to those of antibodies, aptamers have several advantages that could outweigh antibody technology and open new opportunities for better medical and diagnostic solutions. However, the current status of the aptamer technology suffers from several technical limitations that slowdown the progression of novel aptamers into the clinic and makes the business around aptamers challenging.

The Rate of Tau Synthesis is Differentially Regulated During Postnatal Development in Mouse Cerebellum

Abstract1. Tau, which is a microtubule-associated protein, with mRNA targeted to the axon and growth cone, is involved in axonal elongation. During postnatal development in mouse, Tau expression in cerebellar granule cells is reduced after the second postnatal week. The aim of this work was to study the regulation of the rate of the synthesis of Tau protein during the period of granule cell axonal growth in mouse cerebellum.2. We found four [35S]methionine-labeled isoforms of Tau synthesized postnataly. Their levels remain constant from postnatal day 9 to 12 (P9–P12), and decreased by P20.3. The rate of Tau synthesis showed differences with the rate of synthesis of total proteins. They also differ from proteins phosphatases 2A and 2B, both associated with the regulation of Tau function. In addition, the turnover of newly synthesized Tau increased at P20, compared with P9 and P12.4. These results imply a specific developmental regulation of mRNA translation of Tau, and indicate that, after the period of synapse formation is complete, and therefore axonal growth has finished (P20), only a limited number of new Tau molecules are synthesized. This might reflect that, after synapse formation is complete, newly synthesized Tau molecules are not longer needed.

Sperm genome cloning used in biparental bovine embryo reconstruction

The generation of androgenetic haploid embryos enables several haploid blastomeres to be obtained as identical copies of a single spermatozoon genome. In the present study, we compared the developmental ability of bovine androgenetic haploid embryos constructed by different methods, namely IVF and intracytoplasmic sperm injection (ICSI) before and after oocyte enucleation. Once obtained, the blastomeres of these androgenetic haploid embryos were used as male genome donors to reconstruct biparental embryos by fusion with matured oocytes. To verify the cytoplasmic contribution of androgenetic haploid blastomeres, we used spermatozoa incubated previously with exogenous DNA that coded for a green fluorescent protein gene (pCX-EGFP) and the enhanced green fluorescent protein (EGFP)-positive androgenetic haploid blastomeres generated were fused with mature oocytes. Of the reconstructed embryos reaching the cleavage and blastocyst stages, 85.1% and 9.0%, respectively, expressed EGFP (P>0.05). EGFP expression was observed in 100% of reconstructed embryos, with 91.2% exhibiting homogenic expression. To confirm sperm genome incorporation, androgenetic haploid blastomeres generated by ICSI prior to enucleation and using Y chromosome sexed spermatozoa were used for biparental embryo reconstruction. Incorporation of the Y chromosome was confirmed by polymerase chain reaction and fluorescence in situ hybridisation analysis. In conclusion, the results of the present study prove that it is possible to use sperm genome replicates to reconstruct biparental bovine embryos and that it is a highly efficient technique to generate homogeneous transgene-expressing embryos.

Different conformations of phosphatase and tensin homolog, deleted on chromosome 10 (PTEN) protein within the nucleus and cytoplasm of neurons.

PTEN is a critical gene involved in the regulation of many cellular processes. The product of this gene has dual phosphatase activity and is able to dephosphorylate the 5′ end of the phosphatidylinositol (3,4,5)-trisphosphate. Within the cellular nucleus, this protein has been associated with regulation of the expression of many genes, although the mechanism of this regulation remains unclear. In this paper, two specific oligonucleotide aptamers were developed and selected, using the SELEX procedure, according to their ability to detect the PTEN protein in different subcellular compartments of neurons. While one aptamer was able to detect PTEN in the nucleus, the other recognized PTEN in the cytoplasm. The recognition pattern of PTEN by both aptamers was confirmed using antibodies in western blots of the proteins purified from mouse cerebellar homogenates and subcellular fractions. Additionally, we demonstrated that the two aptamers recognized different epitopes of the target peptide. The results presented here could not be fully explained by the canonical phosphatase structure of PTEN, suggesting the existence of different conformations of phosphatase in the nucleus and the cytoplasm.

In Vitro and In Vivo Development of Horse Cloned Embryos Generated with iPSCs, Mesenchymal Stromal Cells and Fetal or Adult Fibroblasts as Nuclear Donors

The demand for equine cloning as a tool to preserve high genetic value is growing worldwide; however, nuclear transfer efficiency is still very low. To address this issue, we first evaluated the effects of time from cell fusion to activation (<1h, n = 1261; 1-2h, n = 1773; 2-3h, n = 1647) on in vitro and in vivo development of equine embryos generated by cloning. Then, we evaluated the effects of using different nuclear donor cell types in two successive experiments: I) induced pluripotent stem cells (iPSCs) vs. adult fibroblasts (AF) fused to ooplasts injected with the pluripotency-inducing genes OCT4, SOX2, MYC and KLF4, vs. AF alone as controls; II) umbilical cord-derived mesenchymal stromal cells (UC-MSCs) vs. fetal fibroblasts derived from an unborn cloned foetus (FF) vs. AF from the original individual. In the first experiment, both blastocyst production and pregnancy rates were higher in the 2-3h group (11.5% and 9.5%, respectively), respect to <1h (5.2% and 2%, respectively) and 1-2h (5.6% and 4.7%, respectively) groups (P<0.05). However, percentages of born foals/pregnancies were similar when intervals of 2-3h (35.2%) or 1-2h (35.7%) were used. In contrast to AF, the iPSCs did not generate any blastocyst-stage embryos. Moreover, injection of oocytes with the pluripotency-inducing genes did not improve blastocyst production nor pregnancy rates respect to AF controls. Finally, higher blastocyst production was obtained using UC-MSC (15.6%) than using FF (8.9%) or AF (9.3%), (P<0.05). Despite pregnancy rates were similar for these 3 groups (17.6%, 18.2% and 22%, respectively), viable foals (two) were obtained only by using FF. In summary, optimum blastocyst production rates can be obtained using a 2-3h interval between cell fusion and activation as well as using UC-MSCs as nuclear donors. Moreover, FF line can improve the efficiency of an inefficient AF line. Overall, 24 healthy foals were obtained from a total of 29 born foals.

Improvement of bovine semen quality by removal of membrane-damaged sperm cells with DNA aptamers and magnetic nanoparticles

In cattle, cryopreservation of semen and sex-sorting kill up to 50% of spermatozoa and decrease the success of assisted insemination (AI). Therefore, significant efforts are being carried out to improve the quality of semen prior to AI. In this work we used the Cell-SELEX technique to select single strand DNA aptamers able to recognize with high affinity and specificity damaged sperm cells generated by heat-treatment. We first isolated aptamers with a conserved two motifs of 6 nucleotides of length that bind to the membrane of heat-treated spermatozoa. Then, we used synthetic biotin-labeled aptamers containing the conserved motif to recognize membrane-damaged cells and separate them from viable cells by the use of avidin-coated superparamagnetic iron oxide nanoparticles (SPION). This procedure improved the quality of semen by significantly increasing the percentage of healthy sperm cells without affecting the rate of blastocyst cleavage. This technique was successfully applied to both unsorted and sex-sorted sperm suspension.

Dystonia in a Patient with Autosomal‐Dominant Progressive External Ophthalmoplegia Type 1 Caused by Mutation in the POLG Gene

Autosomal-dominant progressive external ophthalmoplegia type 1 (adPEO1) is characterized by slowly progressive ophthalmoplegia. It can be caused by mutations in different genes, including the mitochondrial DNA polymerase c (POLG), which results in heterogeneous clinical phenotypes associated with progressive external ophthalmoplegia, including myoclonic epilepsy, parkinsonism, and ataxia (Table 1). Other additional features may include premature ovarian failure and hypogonadism. POLG mutations causing adPEO1 can have both autosomal-dominant or -recessive inheritance traits. Dystonia has been observed in patients with POLG mutations, however, to our knowledge, not in adPEO1. Here, we describe the first adPEO1 patient attributed to a POLG mutation showing dystonia as the presenting and core clinical feature.