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.     

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.     

Hemopoietic and Lymphoid Progenitor Cells in Human Umbilical Cord Blood

Human umbilical cord blood, which in the past was discarded with the placental tissue, provides a convenient source of fetal hemopoietic cells for scientific analysis and clinical use. Cord blood cells are immature compared to analogous populations in adult peripheral blood. Cord blood B lymphocytes display unique phenotypic and functional characteristics. The antigens CD1C, CD38, CD5, and CD23, although normally expressed on only a small percentage of circulating B cells in adults, are highly expressed on cord blood B cells. Recent studies have demonstrated that whereas cord blood B cells are functionally naive, their potential is similar to that of adult B cells if optimal T-cell help is available. Thus, the failure of B-cell responses in cord blood is due to the T cells. The functional abnormalities of T cells from newborns can be summarized as a dominance of the effects of TH0 cells. Thus, the cytokines produced are immunosuppressive rather than mediating helper activity for B cells. NK activity in cord blood is also depressed compared to that in adults. Cord blood is a very rich source of hemopoietic progenitor cells. The spectrum of progenitors shows a predominance of early progenitor cells when compared with bone marrow. These cells provide an alternative source to adult bone marrow for stem cells to use for hemopoietic reconstitution and as targets in the treatment of hereditary deficiencies by gene therapy. These features make cord blood a unique research tool to investigate hemopoietic ontogeny and a unique clinical tool for transplantation.     

Mobility and symmetry in the Fc and pFc' fragments as probed by 1H NMR

The 270 MHz ¹H NMR spectra of rabbit Fc and pFc′ fragments appear well resolved when compared to the spectra of other proteins of similar molecular weight. This is interpreted as evidence for substantial segmental flexibility in these fragments. This mobility can be monitored using two special NMR pulse sequences which exploit either the multiplet structure (spin coupling) or the differential linewidths of resonances. Several aromatic residues with mobility independent of the rest of the protein could be detected by these techniques.The titration behaviour of the histidine residues of pFc′ indicates that this fragment possesses a longitudinal C-2 symmetry axis. Of the three pairs of histidines in pFc′, two titrate with pK_a values of 6.8 and 7.3, while the remaining pair remains protonated over the pH range 5.2–8.2 suggesting that these residues are not readily accessible to the solvent. The observation of five pairs of histidine C-2 proton resonances in the Fc indicates that the symmetry axis is retained in this larger fragment.Cleavage of the inter-heavy chain disulphide bond in the Fc fragment has little effect on its NMR spectrum. We find from this that the reduction in complement binding efficiency associated with this cleavage is not a direct consequence of a concomitant large structural change in the Fc fragment.     

The Role of Trait Anxiety in Nicotine Dependence1

Studies point to an association between anxiety and smoking. However, the mechanisms linking trait anxiety and nicotine dependence have not been evaluated fully. Potential mediators include self-medication variables (e.g., use of nicotine to manage anxiety) and cognitive variables (e.g., lower levels of self-efficacy). The present study explored these mechanisms in a sample of 352 male and female smokers. The results showed that trait anxiety correlated significantly with negative affect smoking (r= .29, p= .0001), stimulation smoking (r=.15, p = .007), and nicotine dependence (r= .20, p= .0003). Trait anxiety also correlated significantly with self-efficacy (r =-.22, p = .0003). Regression analyses revealed that trait anxiety predicted nicotine dependence after controlling for depression, education, race, age, and marital status (R²= .09, p = .0001). Path modeling indicated that both negative affect smoking and quitting self-efficacy mediated the relationship between trait anxiety and nicotine dependence. Interventions that emphasize the management of anxious mood and quitting confidence may benefit anxious smokers.     

Thymic Medulla Epithelial Cells Acquire Specific Markers by Post-Mitotic Maturation

The development of thymocyte subsets and of the thymic epithelium in SCID and RAG-2^-/– mice was monitored after normal bone-marrow-cell transfer. The kinetics of thymic reconstitution and their relationships with cell proliferation were investigated by using bromodeoxyuridine to detect DNA-synthesizing cells among lymphoid cells by 3-color flow cytometry, and in epithelial compartments by staining frozen sections. Thymocytes started to express CD8 and CD4 10 days after transfer, simultaneously with extensive proliferation. The first mature CD4⁺ single-positive cells were generated, from resting CD4⁺CD8⁺ cells after day 15. During this day 10–15 period, many epithelial cells positive for cortexspecific or panepithelial markers were labeled with BrdUrd after pulse-injection. Organized medullary epithelium also developed after day,15, that is, synchronously with the appearance of mature thymocytes, but medullary cells were never found BrdUrd⁺. These results suggest that, in these models, the reconstitution of the thymic epithelial network proceeds through expansion of preexisting cortical or undifferentiated cells and by later maturation (acquisition of specific markers) of medullary cells. This last process is dependent of the presence of mature thymocytes.     

Induction by sphingomyelinase of shiga toxin receptor and shiga toxin 2 sensitivity in human microvascular endothelial cells

Shiga toxin-producing enterohemorrhagic Escherichia coli is the major cause of acute renal failure in young children. The interaction of Shiga toxins 1 and 2 (Stx1 and Stx2) with endothelial cells is an important step in the renal coagulation and thrombosis observed in hemolytic uremic syndrome. Previous studies have shown that bacterial lipopolysaccharide and host cytokines slowly sensitize endothelial cells to Shiga toxins. In the present study, bacterial neutral sphingomyelinase (SMase) rapidly (1 h) sensitized human dermal microvascular endothelial cells (HDMEC) to the cytotoxic action of Stx2. Exposure of endothelial cells to neutral SMase (0.067 U/ml) caused a rapid increase of intracellular ceramide that persisted for hours. Closely following the change in ceramide level was an increase in the expression of globotriaosylceramide (Gb3), the receptor for Stx2. A rapid increase was also observed in the mRNA for ceramide:glucosyltransferase (CGT), the first of three glycosyltransferase enzymes of the Gb3 biosynthetic pathway. The product of CGT (glucosylceramide) was also increased. In contrast, mRNA for the third enzyme of the pathway, Gb3 synthase, was constitutively produced and was not influenced by SMase treatment of HDMEC. These results describe a rapid response mechanism by which extracellular neutral SMase derived from either bacteria or eukaryotic cells may signal endothelial cells to become sensitive to Shiga toxins.