Reprogramming alternative pre-messenger RNA splicing through the use of protein-binding antisense oligonucleotides

Alternative pre-messenger RNA splicing is a major contributor to proteomic diversity in higher eukaryotes and represents a key step in the control of protein function in a large variety of biological systems. As a means of artificially altering splice site choice, we have investigated the impact of positioning proteins in the vicinity of 5' splice sites. We find that a recombinant GST-MS2 protein interferes with 5' splice site use, most efficiently when it binds upstream of that site. To broaden the use of proteins as steric inhibitors of splicing, we have tested the activity of antisense oligonucleotides carrying binding sites for the heterogeneous nuclear ribonucleoprotein A1/A2 proteins. In a HeLa cell extract, tailed oligonucleotides complementary to exonic sequences elicit strong shifts in 5' splice site selection. In four different human cell lines, an interfering oligonucleotide carrying A1/A2 binding sites also shifted the alternative splicing of the Bcl-x pre-mRNA more efficiently than oligonucleotides acting through duplex formation only. The use of protein-binding oligonucleotides that interfere with U1 small nuclear ribonucleoprotein binding therefore represents a novel and powerful approach to control splice site selection in cells.     

Receptor autoradiography as mean to explore the possible functional relevance of neuropeptides: focus on new agonists and antagonists to study natriuretic peptides, neuropeptide Y and calcitonin gene-related peptides

Over the past 20 years, receptor autoradiography has proven most useful to provide clues as to the role of various families of peptides expressed in the brain. Early on, we used this method to investigate the possible roles of various brain peptides. Natriuretic peptide (NP), neuropeptide Y (NPY) and calcitonin (CT) peptide families are widely distributed in the peripheral and central nervous system and induced multiple biological effects by activating plasma membrane receptor proteins. The NP family includes atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). The NPY family is composed of at least three peptides NPY, peptide YY (PYY) and the pancreatic polypeptides (PPs). The CT family includes CT, calcitonin gene-related peptide (CGRP), amylin (AMY), adrenomedullin (AM) and two newly isolated peptides, intermedin and calcitonin receptor-stimulating peptide (CRSP). Using quantitative receptor autoradiography as well as selective agonists and antagonists for each peptide family, in vivo and in vitro assays revealed complex pharmacological responses and radioligand binding profile. The existence of heterogeneous populations of NP, NPY and CT/CGRP receptors has been confirmed by cloning. Three NP receptors have been cloned. One is a single-transmembrane clearance receptor (NPR-C) while the other two known as CG-A (or NPR-A) and CG-B (or NPR-B) are coupled to guanylate cyclase. Five NPY receptors have been cloned designated as Y(1), Y(2), Y(4), Y(5) and y(6). All NPY receptors belong to the seven-transmembrane G-protein coupled receptors family (GPCRs; subfamily type I). CGRP, AMY and AM receptors are complexes which include a GPCR (the CT receptor or CTR and calcitonin receptor-like receptor or CRLR) and a single-transmembrane domain protein known as receptor-activity-modifying-proteins (RAMPs) as well as an intracellular protein named receptor-component-protein (RCP). We review here tools that are currently available in order to target each NP, NPY and CT/CGRP receptor subtype and establish their respective pathophysiological relevance.     

Pamidronate: Treatment for severe hypercalcemia in neonatal subcutaneous fat necrosis

BACKGROUND: Subcutaneous fat necrosis (SCFN) of the newborn is an uncommon disorder that occurs in the first weeks of life after foetal distress. It can be complicated by potentially life-threatening hypercalcemia. Treatments of hypercalcemia have included hydration, furosemide and corticosteroids. Only one report has described the use of intravenous bisphosphonates for this condition. We propose that pamidronate could be the first line therapy for severe hypercalcemia in SCFN. PATIENTS AND RESULTS: Four newborns presented between 2001 and 2004 with SCFN complicated by severe hypercalcemia. At diagnosis, ionized calcium levels were higher than 1.4 mmol/l and were associated with high urinary calcium/creatinine ratios and high 1,25-dihydroxyvitamin D levels. Despite treatment with IV fluids, low calcium diet and furosemide, calcium levels remained high. The patients were given 3-4 doses (0.25-0.50 mg/kg/dose) of pamidronate. Urinary calcium/creatinine ratios and calcium levels decreased within 48-96 h. 1,25-dihydroxyvitamin D levels normalized with resolution of the skin lesions. No persistent nephrocalcinosis was observed. CONCLUSION: Pamidronate is effective, well-tolerated in the short-term and obviates the need for prolonged treatment with furosemide and corticosteroids. To prevent nephrocalcinosis, pamidronate might be considered as first line treatment for severe hypercalcemia in SCFN.     

Organ culture of adult mouse intestine. III. Behavior of the proteins, DNA content and brush border membrane enzymatic activities.

The proteins, the DNA content and the brush border membrane enzyme activities of adult mouse intestinal explants have been measured during a 24-hr organ culture. These activities were not modified in comparison with the controls at the beginning of the culture. The activity of the enzymes secreted in the medium during the 24-hr culture was equal to the activity present in the explants at the start of the culture. These results show that several metabolic functions of the intestine are fully preserved in organ culture.     

Elevated cyclic AMP levels fail to inhibit extracellular calcium dependent histamine secretion from rat mast cells

Rat mast cells preincubated with chelating agent and bathed in calcium-free medium fail to release histamine when stimulated by compound 48/80 and show elevated levels of cyclic AMP. When calcium is added to the bathing medium, these cells undergo a prompt secretory response yet cyclic AMP levels do not fall. It is suggested that the level of cyclic AMP may not regulate the change in membrane permeability believed produced by compound 48/80.     

Hypoxia tolerance in Atlantic cod

Oxygen saturation levels that killed 50 and 5% of cod Gadus morhua over 96 h averaged 21·2 and 27·7%, respectively. No fish survived at 10% saturation and only a few survived at 16% saturation, whereas no mortality occurred at 34 and 40% oxygen saturation. Since metabolic rate and oxygen consumption increase with increasing temperature, we hypothesized that cod would be less tolerant to hypoxic conditions at 6 than at 2° C. However, temperature (2 and 6° C) had no measurable impact on cod survival. Small (mean & S.D.; 45·2 ± 4·2 cm) and large (57·5 ± 3·8 cm) cod had the same tolerance to hypoxia. At the end of the experiments, hypoxia had a significant effect on blood haematocrit, mean cellular haemoglobin content, liver lactate, plasma glucose and plasma lactate, but accounted for only a small fraction (< 10%) of the variation, except for plasma lactate which exhibited a strong response with concentrations increasing progressively with decreasing levels of oxygen saturation. Temperature had a significant effect on most variates in normoxia and hypoxia. Variates also affected by oxygen level showed significant interactions between oxygen and size or temperature effects. However, these interactions accounted for only a small proportion of the variation. Physiological parameters indicated that extending the duration of our tests beyond 96 h would not have changed our estimates of the lethal thresholds. Hypoxic conditions are a permanent feature of the deep waters of the Gulf of St Lawrence. This study shows that a significant portion of the benthic habitats in the Gulf are uninhabitable for cod which would be expected to avoid waters below 28% oxygen saturation.     

Organ culture of adult mouse intestine

Summary Explants of adult mouse intestine have been maintained in organ culture for 24 to 48 hr. The best results have been obtained with a mixture of DMEM-HEPES medium and NCTC-135 enriched with 10% fetal bovine serum. The morphology of the mucosa is well preserved at the light electron microscopic level: absorbing cells exhibit an increase in secondary lysosomes; goblet cells and Paneth cells remain active; numerous mitoses are observed in the crypts; and vigorous re-epithelization takes place on the margin of the explants. This work was supported by a research grant from the Medical Research Council of Canada (to J. S. H.) and partly by the F.C.A.C. Québec (to J. S. H.).     

Valid oxygen uptake measurements: using high r2 values with good intentions can bias upward the determination of standard metabolic rate

This analysis shows good intentions in the selection of valid and precise oxygen uptake (O₂) measurements by retaining only slopes of declining dissolved oxygen level in a respirometer that have very high values of the coefficient of determination, r², are not always successful at excluding nonlinear slopes. Much worse, by potentially removing linear slopes that have low r² only because of a low signal-to-noise ratio, this procedure can overestimate the calculation of standard metabolic rate (SMR) of the fish. To remedy this possibility, a few simple diagnostic tools are demonstrated to assess the appropriateness of a given minimum acceptable r², such as calculating the proportion of rejected O₂ determinations, producing a histogram of the r² values and a plot of r² as a function of O₂. The authors offer solutions for cases when many linear slopes have low r². The least satisfactory but easiest to implement is lowering the minimum acceptable r². More satisfactory solutions involve processing (smoothing) the raw signal of dissolved oxygen as a function of time to improve the signal-to-noise ratio and the r²s.