Detection of a 15q deletion in a child with Angelman syndrome by cytogenetic analysis and flow cytometry

A proximal 15q deletion, del(15) (q11:q13), was detected in a child with Angelman syndrome by cytogenetic analysis of peripheral lymphocytes. The chromosomes of both parents appeared normal. Flow karyotype analysis carried out on lymphoblastoid cell lines derived from the child and her parents confirmed the presence of a de novo 15 deletion. The estimated size of the deleted segment ranged from 6.1-9.5% of chromosome 15 (approximately 6-9.3 million base pairs). The parental origin of the deleted chromosome could not be resolved by flow cytometry, but cytogenetic evidence suggested that it was derived from the smaller chromosome 15 homologue in the mother.     

Elastin point mutations cause an obstructive vascular disease, supravalvular aortic stenosis

Supravalvular aortic stenosis (SVAS) is an inherited obstructive vascular disease that affects the aorta, carotid, coronary and pulmonary arteries. Previous molecular genetic data have led to the hypothesis that SVAS results from mutations in the elastin gene, ELN. In these studies, the disease phenotype was linked to gross DNA rearrangements (35 and 85 kb deletions and a translocation) in three SVAS families. However, gross rearrangements of ELN have not been identified in most cases of autosomal dominant SVAS. To define the spectrum of ELN mutations responsible for this disorder, we refined the genomic structure of human ELN and used this information in mutational analyses. ELN point mutations co-segregate with the disease in four familial cases and are associated with SVAS in three sporadic cases. Two of the mutations are nonsense, one is a single base pair deletion and four are splice site mutations. In one sporadic case, the mutation arose de novo. These data demonstrate that point mutations of ELN cause autosomal dominant SVAS.      

Comparison of DNA- and RNA-based methods for detection of truncating BRCA1 mutations

A number of methods are used for mutational analysis of BRCA1, a large multi-exon gene. A comparison was made of five methods to detect mutations generating premature stop codons that are predicted to result in synthesis of a truncated protein in BRCA1. These included four DNA-based methods: two-dimensional gene scanning (TDGS), denaturing high performance liquid chromatography (DHPLC), enzymatic mutation detection (EMD), and single strand conformation polymorphism analysis (SSCP) and an RNA/DNA-based protein truncation test (PTT) with and without complementary 5' sequencing. DNA and RNA samples isolated from 21 coded lymphoblastoid cell line samples were tested. These specimens had previously been analyzed by direct automated DNA sequencing, considered to be the optimum method for mutation detection. The set of 21 cell lines included 14 samples with 13 unique frameshift or nonsense mutations, three samples with two unique splice site mutations, and four samples without deleterious mutations. The present study focused on the detection of protein-truncating mutations, those that have been reported most often to be disease-causing alterations that segregate with cancer in families. PTT with complementary 5' sequencing correctly identified all 15 deleterious mutations. Not surprisingly, the DNA-based techniques did not detect a deletion of exon 22. EMD and DHPLC identified all of the mutations with the exception of the exon 22 deletion. Two mutations were initially missed by TDGS, but could be detected after slight changes in the test design, and five truncating mutations were missed by SSCP. It will continue to be important to use complementary methods for mutational analysis.     

A Comparative Analysis of the Murine Thymic Microenvironment in Normal,Autoimmune, and Immunodeficiency States

It is widely accepted that the thymic microenvironment regulates normal thymopoiesis through a highly coordinated and complex series of cellular and cytokine interactions. A direct corollary of this is that abnormalities within the microenvironment could be of etiologic significance in T-cell-based diseases. Our laboratory has developed a large panel of monoclonal antibodies (mAbs) that react specifically with epithelial or nonepithelial markers in the thymus. We have taken advantage of these reagents to characterize the thymic microenvironment of several genetic strains of mice, including BALB/cJ, C57BL/6J, NZB/BlnJ, SM/J, NOD/Ltz, NOD/Ltz-scid/sz, C57BL/6J-Hcph m^e/Hcph m^e, and ALY/NscJcl-aly/aly mice, and littermate control animals. We report herein that control mice, including strains of several backgrounds, have a very consistent phenotypic profile with this panel of monoclonal antibodies, including reactivity with thymic epithelial cells in the cortex, the medulla and the corticomedullary junction, and the extracellular matrix. In contrast, the disease-prone strains studied have unique, abnormal staining of thymic cortex and medulla at both the structural and cellular levels. These phenotypic data suggest that abnormalities in interactions between developing thymocytes and stromal cells characterize disease-prone mice.     

Substrate-charge dependence of stoichiometry shows membrane potential is the driving force for proton-peptide cotransport in rat renal cortex

The proton dependence of the transport of three labelled, hydrolysis-resistant synthetic dipeptides carrying a net charge of –1, 0 or +1 has been investigated in a brush border membrane vesicle preparation obtained from rat renal cortex. Cross-inhibition studies are consistent with the transport of all peptides studied being through a single system. The extent and time course of uptake in response to an inwardly directed electrochemical gradient of protons differed for each peptide. For the cationic peptide D-Phe-L-Lys this gradient did not stimulate the initial rate of uptake, while for the neutral dipeptide D-Phe-L-Ala and the anionic peptide D-Phe-L-Glu stimulation was observed. However, the effect on D-Phe-L-Glu was more marked than that on D-Phe-L-Ala and the proton activation differed for these two peptides. The calculated Hill coefficients for the two proton-dependent peptides were 1.14±0.16 and 2.15±0.10 for D-Phe-L-Ala and D-Phe-L-Glu, respectively, providing evidence that the stoichiometry of proton: peptide cotransport is different for each peptide (01, 11 and 21 for D-Phe-L-Lys, D-Phe-L-Ala and D-Phe-L-Glu respectively); studies on energetics are compatible with this conclusion. The physiological and molecular implications of this model are discussed, as are the applicability of the conclusions to secondary active transport systems more generally.     

Contextual modulation of synchronization to random dots in the cat visual cortex

Synchronization of neuronal activity has been proposed as a binding mechanism for integration of image properties into one coherent percept. In the present study, we investigated the contextual modulation of synchronization to random dot patterns. Coherent motion of random dots evoked well synchronized responses in area 17 of anaesthetized cats when the stimulus was presented in the compound receptive field of recorded sites. Gradually changing the directional coherence of random dots in the surround while maintaining fully coherent motion of the stimulus in the receptive field significantly suppressed synchronization of neuronal activity for some stimulus conditions. However, usually one or two peaks of increased synchronization were found in the surround coherence tuning curves with low (8–12%) and/or moderate (25–50%) coherence in the surround. At the population level, synchronization was significantly depressed with incoherent motion in the receptive field and when both the surround and the receptive field were jointly stimulated with 0% coherence. The intriguing finding was the discovery of two distinct groups of cells with opposite synchronization changes dependent on the presence or absence of significant synchronization in their spontaneous activity. The latter group of neurons showed peaks of increased synchronization with lower surround coherence, thus probably being more sensitive to the direction of the surround motion. Overall, our findings support the notion that binding of stimulus properties can be achieved by synchronized activity of cortical cells. However, our findings go further than the original hypothesis of feature binding by synchrony to show that synchronization of cortical activity may be directly related to the decision making processes, which in turn are related to the threshold of perception of coherent motion.     

Serological responses to the B subunit of Shiga-like toxin 1 and its peptide fragments indicate that the B subunit is a vaccine candidate to counter action of the toxin.

The B subunit of Shiga toxin and Shiga-like toxin (SLT-1) and its fragments are potentially immunogenic and may generate protective humoral responses against the action of these toxins. We have analyzed the antibody response of rabbits immunized with pure B subunit of SLT-1 or synthetic fragments of the subunit. The immune response to the native B subunit was found to be largely directed at conformational epitopes. More importantly, rabbits immunized with the B subunit were protected from a lethal challenge with SLT-1, indicating that the B subunit represents an excellent vaccine candidate to counter the effects of Shiga toxin and SLT-1 in humans. Polyclonal antibodies against a synthetic peptide corresponding to residues 28 to 40 of the B subunit neutralized the cytotoxicity of SLT-1 towards Vero cells. This region is thus exposed in the native state of the B subunit. The sequence specificity of other antipeptide antisera also provides clues to the state of folding and assembly of the B subunit. Antisera to synthetic peptides representing the N- and C-terminal regions of the SLT-1 B subunit did not cross-react with native B subunit but strongly recognized denatured forms of the protein. Finally, the monoclonal antibody 13C4 was shown to bind to a discontinuous epitope expressed only on the native form of the protein. These immunological reagents can be used to probe the conformational state of the B subunit and the holotoxin as it relates to their functional properties.     

Mutations in Emery-Dreifuss muscular dystrophy and their effects on emerin protein expression

Seventeen families with Emery-Dreifuss muscular dystrophy (EDMD) have been studied both by DNA sequencing and by emerin protein expression. Fourteen had mutations in the X-linked emerin gene, while three showed evidence of autosomal inheritance. Twelve of the 14 emerin mutations caused early termination of translation. An in-frame deletion of six amino acids from the C-terminal transmembrane helix caused almost complete absence of emerin from muscle with no localization to the nuclear membrane, although mRNA levels were normal. This shows that mutant emerin proteins are unstable if they are unable to integrate into a membrane. A 22 bp deletion in the promoter region was expected to result in reduced emerin production, but normal amounts of emerin of normal size were found in leucocytes and lymphoblastoid cell lines. This shows that DNA analysis is necessary to exclude emerin mutations in suspected X-linked EDMD. Emerin levels in female carriers often deviated from the expected 50% and this was due, in at least two families, to skewed emerin mRNA expression from the normal and mutated alleles. In one family with a novel deletion of the last three exons of the emerin gene, a carrier had a cardiomyopathy and very low emerin levels (<5% of normal) due to skewed X-inactivation. In the three autosomal cases of EDMD, emerin was normal on western blots of blood cells, which suggests that autosomal EDMD is not caused by indirect reduction of emerin levels.