Wnt/ß-catenin pathway and cell adhesion deregulation in CSDE1-related intellectual disability and autism spectrum disorders.

Among the genetic factors playing a key role in the etiology of intellectual disabilities (IDs) and autism spectrum disorders (ASDs), several encode RNA-binding proteins (RBPs). In this study, we deciphered the molecular and cellular bases of ID-ASD in a patient followed from birth to the age of 21, in whom we identified a de novo CSDE1 (Cold Shock Domain-containing E1) nonsense variation. CSDE1 encodes an RBP that regulates multiple cellular pathways by monitoring the translation and abundance of target transcripts. Analyses performed on the patient's primary fibroblasts showed that the identified CSDE1 variation leads to haploinsufficiency. We identified through RNA-seq assays the Wnt/ß-catenin signaling and cellular adhesion as two major deregulated pathways. These results were further confirmed by functional studies involving Wnt-specific luciferase and substrate adhesion assays. Additional data support a disease model involving APC Down-Regulated-1 (APCDD1) and cadherin-2 (CDH2), two components of the Wnt/ß-catenin pathway, CDH2 being also pivotal for cellular adhesion. Our study, which relies on both the deep phenotyping and long-term follow-up of a patient with CSDE1 haploinsufficiency and on ex vivo studies, sheds new light on the CSDE1-dependent deregulated pathways in ID-ASD.

Cloning and characterisation of a gene from Plasmodium vivax and P. knowlesi: homology with valine-tRNA synthetase.

We have previously described a lambdagt11 clone detected by immune screening with a monoclonal antibody (mAb) A12. This mAb is capable of completely blocking Plasmodium vivax transmission in the mosquito vector. An epitope recognised by A12 was mapped to six amino acids (aa) within the translated sequence of this clone. Here, we describe the complete sequence of the gene within which we mapped this epitope. Surprisingly, the translated sequence of the full-length open reading frame shows homology with that of valine-tRNA synthetases (Val-tRS) from other organisms. DNA cross-hybridisation with several of these species was observed by Southern blot. In addition, the corresponding gene has been obtained from the closely related simian malaria parasite, P. knowlesi. The two aa sequences show 66% identity and yet are very divergent from other Val-tRS sequences, apart from conserved blocks related to functional activity. Multiple sequence alignments reflect this dichotomy, as do predicted differences in antigenicity.

Transmission blocking immunity in Plasmodium vivax malaria: antibodies raised against a peptide block parasite development in the mosquito vector.

One approach towards the development of a vaccine against malaria is to immunize against the parasite sexual stages that mediate transmission of the parasite from man to mosquito. Antibodies against these stages, ingested with the blood meal, inhibit the parasite development in the mosquito vector, constituting "transmission blocking immunity." Most epitopes involved in transmission-blocking immunity depend on the tertiary conformational structure of surface antigens. However, one of the transmission-blocking monoclonal antibodies we have raised against Plasmodium vivax reacts with a linear epitope on both asexual stages and gametes. This monoclonal antibody (A12) is capable of totally blocking development of the parasite in the mosquito host when tested in membrane feeding assays with gametocytes from P. vivax-infected patients. Immune screening of a P. vivax lambda gt11 genomic expression library with A12 led to the isolation of a clone to which was mapped the six-amino acid epitope recognized by A12. Antisera raised in mice against a 12-mer synthetic peptide containing this epitope coupled to bovine serum albumin not only had high titers of antipeptide antibodies as measured by enzyme-linked immunosorbent assay, but in addition recognized the same 24- and 57-kD parasite components as A12 on Western blots and reacted with the parasite by immunofluorescence. When tested in membrane feeding assays, these antibodies have significant suppressive effects on parasite development in the mosquito.

Plasmodium vivax:recombination between potential allelic types of the merozoite surface protein MSP1 in parasites isolated from patients.

The merozoite surface protein MSP1, which is one of the most promising candidates for a malaria vaccine directed against erythrocytic stages, has been shown to be polymorphic in different malarial species. Characterization of the Plasmodium vivax MSP1 gene (Pv200) in two strains (Belem and Salvador-1) revealed the existence of several polymorphic regions. One of these regions has been examined here in primary parasite isolates obtained from patients in Sri Lanka. Oligonucleotide primers hybridizing to conserved parts of the gene on either side of a polymorphic region were used to amplify DNA from 22 isolates. Sequence analysis of the amplified portion of the MSP1 gene in five patients showed the existence of three types of polymorphic regions. Two were almost identical either to that of the Belem or to that of the Salvador-1 strain. The third polymorphic type appeared to have resulted from recombination between the two others. This recombination event took place inside a repeated part of the sequence.

Primary structure of the merozoite surface antigen 1 of Plasmodium vivax reveals sequences conserved between different Plasmodium species.

Merozoite surface antigen 1 (MSA1) of several species of plasmodia has been shown to be a promising candidate for a vaccine directed against the asexual blood stages of malaria. We report the cloning and characterization of the MSA1 gene of the human malaria parasite Plasmodium vivax. This gene, which we call Pv200, encodes a polypeptide of 1726 amino acids and displays features described for MSA1 genes of other species, such as signal peptide and anchoring sequences, conserved cysteine residues, number of potential N-glycosylation sites, and repeats consisting here of 23 glutamine residues in a row. When the nucleotide and deduced amino acid sequences of the MSA1 of P. vivax are compared to those of another human malaria parasite, Plasmodium falciparum, and to those of the rodent parasite Plasmodium yoelii, 10 regions of high amino acid similarity are observed despite the very different dG + dC contents of the corresponding genes. All of the interspecies conserved regions reside within the conserved or semiconserved blocks delimited by the sequences of different alleles of the MSA1 gene of P. falciparum.

Plasmodium vivax: karyotype polymorphism of field isolates.

Pulse-field gradient electrophoresis (PFG) has been applied to the karyotype analysis of Plasmodium vivax isolates obtained directly from infected patients in Sri Lanka. Detection of separated chromosomes was performed either by ethidium bromide staining of gels or by hybridization with a telomer specific probe. Each of the 15 different isolates examined exhibited a different chromosome migration pattern, indicating that a high level of polymorphism prevailed in wild populations of P. vivax. Chromosome size variation was further confirmed using a P. vivax chromosome-specific probe which also demonstrated that, in each isolate, the parasite population appeared to be homogeneous. These observations were made directly on parasites from infected blood, without the necessity for culture amplification, indicating that PFG can be used on a large scale for the epidemiological analysis of wild parasite populations.

Plasmodium vivax: cloning and expression of a major blood-stage surface antigen.

Plasmodium vivax is a highly prevalent malaria pathogen of man; the following report is the first to describe the cloning and expression of a major asexual erythrocytic stage antigen of this species. The screening of a genomic DNA expression library with a monoclonal antibody directed against a 200-kDa surface component (Pv200) of the more mature schizonts of P. vivax led to the selection of a recombinant bacterial clone which produced a fusion protein. Mouse and rabbit immune sera raised against the purified fusion protein recognized the 200-kDa parasite antigen on Western blots and reacted with the surface of segmenters by immunofluorescence. Sequencing of the 1.9-kb P. vivax DNA insert coding for this fusion protein revealed a 45-47% homology at the nucleotide level with the P. falciparum gene of a parasite surface antigen, Pf195, which has been shown to be a promising candidate for a malaria vaccine in primates and in man.

Coronary vasoactivity of adenosine in the conscious dog.

Intracoronary adenosine infusions in conscious dogs produced half-maximal coronary vasodilation at 0.57 +/- 0.18 (SD) microns and at 1.01 +/- 0.25 microns in open-chest dogs. In both preparations, adenosine at concentrations in the range found in cardiac muscle by direct analysis produced coronary vasodilation equal to that attained during a maximum reactive hyperemic response. The quantitative structure-activity relationship technique was applied to data on the coronary vasoactivity of 68 adenosine analogs to identify the chemical features of this molecule that determine its vasoactivity. These are: (1) the size of the purine base; (2) the inductive effect of C-2 substituent; (3) the electron-withdrawing effect of the C-6 substituent; (4) the glycosylic torsion angle; (5) the ability of the C-2' and C-3' hydroxyls to participate in hydrogen bonding; (7) the absence of sterically hindering groups in the vicinity of C-2' and, more importantly, C-3'; and (8) the inductive effect of the C-5' substituent. The hydrophobicity of these analogs did not correlate with vasoactivity, suggesting that the hydrophilicity of the ribose moiety overshadows any hydrophobic influence of the very weakly aromatic purine base.

An implantable semiconductor beta-radiation detector.

An implantable beta-radiation detector suitable for the measurement of reginal blood flow in the experimental animal by the indicator clearance principle is described. A lithium-drifted silicon diode encapsulated in a stainless steel case is sutured over the site of interest. A suitable beta-emitting isotope, such as 85Kr in saline solution, is injected into the arterial supply and its calibrated against a mechanical system and showed excellent agreement up to 600 ml/100 g per min. At very high rates beyond the physiological range, flow was underestimated by a maximum of 10%. In vivo comparisons of myocardial blood flow assessed by the beta detector did not agree well with estimates of flow from a precordial counter or by the microsphere technique. Possible reasons are spatial heterogeneity of regional myocardial blood flow, the greatly different masses of tissue involved, or our inability to achieve sufficient numbers of spheres for accuracy in a 50-mg mass of tissue. The unit was still functional after 50 days in a chronic animal.

Phasic right coronary artery blood flow in conscious dogs with normal and elevated right ventricular pressures.

We studied phasic right coronary blood flow in well trained normal dogs and dogs with pulmonic stenosis. We installed electromagnetic flow transducers and pressure tubes under anesthesia to monitor right coronary blood flow, cardiac output, central aortic blood pressure, and right ventribular pressure. In normotensive dogs, systolic flow amplitude equaled early diastolic flow levels. The ratio of systolic to diastolic flow at rest was substantially greater in the right coronary bed (36+/-1.3%) than in the left circumflex bed (13+/-3.6%). Right diastolid flow runoff, including the cove late in diastole, resembled left circumflex runoff. Blood flow to the normotensive right (37+/-1.1 ml/min 100(-1) g) and the left (35+/-1.0 ml/min(-1) g) ventricular myocardium indicated equal perfusion of both cardiac walls. Throttling of systolic flow was related directly to the right ventricular systolic pressure level in the dogs with pulmonic stenosis. Retrograde systolic flow occurred in severe right ventricular hypertension. The late diastolic runoff pattern in dogs with pulmonic stenosis appeared the same as for the normotensive dogs. We obtained systolic to diastolic flow ratios of 1/3 the value of normotensive hearts in high and severe pulmonic hypertension. Electrocardiograms and studies of pathology suggested restricted blood flow to the inner layers of the right myocardium in the dogs with severe and high right ventricular hypertension. Normotensive and hypertensive peak hyperemic flow responses were similar, except for an increased magnitude of diastolic flow, with proportionately less systolic flow in hypertensive states.

Evidence for an adenosine receptor on the surface of dog coronary myocytes.

Adenosine and theophylline were linked covalently to oxidized stachyose to produce compounds too large to penetrate cell membranes. These compounds were used in two conscious and six open-chest anesthetized dogs to test the hypothesis that there is an adenosine receptor on the surface of the coronary myocyte. Intracoronary infusions of the adenosine derivative produced dose-dependent coronary vasodilation which was antagonized by theophylline; two types of theophylline derivative antagonized the coronary vasodilatory action of adenosine. Although these results show that both adenosine and theophylline exert their coronary effects at the surface of the smooth muscle cells, this evidence does not establish that they are competing for a common receptor.