Rapid DNA haplotyping using a multiplex heteroduplex approach: Application to duchenne muscular dystrophy carrier testing

A new strategy has been developed for rapid haplotype analysis based on an initial multiplex amplification of several polymorphic sites, followed by heteroduplex detection. Heteroduplexes formed between two different alleles are detected because they migrate differently than the corresponding homoduplexes in Hydrolink-MDE gel. This simple, rapid method does not depend on specific sequences such as restriction enzyme sites or CA boxes and does not require the use of isotope. This approach has been tested using commonly occurring polymorphisms spanning the dystrophin gene as a model. We describe the use of the method to assign the carrier status of females in Duchenne muscular dystrophy (DMD) pedigrees. The method may be used for other genetic diseases when mutations are unknown or there are few dinucleotide markers in the gene proximity, and for the identification of haplotype backgrounds of mutant alleles. © 1995 Wiley-Liss, Inc.     

Nucleotide sequence of the coat protein gene of a necrotic strain of potato virus Y from New Zealand

Summary The sequence of the 3-terminal 1,134 nucleotides of the genome of a New Zealand isolate of a necrotic strain of potato virus Y (PVY^N) has been determined. This sequence contains one large open reading frame of 796 nucleotides, the start of which was not identified, which is capable of encoding a protein of 264 amino acid residues with a molecular weight of 29,631. Comparison of the amino acid sequence with a published coat protein sequence of another strain, PVY-D, and with the amino acid sequence deduced from PeMV cDNA sequence data, confirms that the 3 cistron encodes the viral coat protein in PVY^N. Adjacent to the 3 end of the coding region there is an untranslatable sequence of 326 nucleotides terminating in a polyadenylate tract. An alignment of the PVY^N amino acid sequence with the coat protein sequences of six other potyviruses revealed significant sequence similarities in the internal and carboxy terminal regions. Much amino acid sequence similarity was found between PVY^N, PVY-D, and PeMV (91–93%), suggesting that PeMV should be regarded as a PVY strain. An analysis of the 3-untranslated region of the six potyviruses revealed PVY^N and PeMV as the only viruses displaying sequence similarity in this region. The 3-untranslated sequences of PVY^N and PeMV were further examined for secondary structure.     

The A427 anchorage-independence assay as a screening tool to identify potential chemopreventive agents

An in vitro model for screening potential chemopreventive agents using inhibition of anchorage-independent growth of a human lung tumor cell line, A427, is described. A427 cells were selected for the model development, as they are known to be tumorigenic in animals, can grow in soft agarose, and their growth can be inhibited by a well-known chemopreventive agent, 13-cis-retinoic acid. Cells are plated on agarose, allowed to develop colonies for 28 days, the stained colonies are enumerated, and the inhibition of spontaneous colony formation measured. A cytotoxicity test is used concurrently with anchorage independent assay for measuring the relative survival of cells to ensure that any observed inhibition of anchorage independent growth is due to the biological activity of the chemopreventive agents, as it uses human cells as substrates rendering the efficacy data feasible for direct extrapolation to humans.     

Mycoplasma pulmonis Inhibits Electrogenic Ion Transport across Murine Tracheal Epithelial Cell Monolayers

Murine chronic respiratory disease is characterized by persistent colonization of tracheal and bronchial epithelial cell surfaces by Mycoplasma pulmonis, submucosal and intraluminal immune and inflammatory cells, and altered airway activity. To determine the direct effect of M. pulmonis upon transepithelial ion transport in the absence of immune and inflammatory cell responses, primary mouse tracheal epithelial cell monolayers (MTEs) were apically infected and assayed in Ussing chambers. M. pulmonis-infected MTEs, but not those infected with a nonmurine mycoplasma, demonstrated reductions in amiloride-sensitive Na⁺ absorption, cyclic AMP, and cholinergic-stimulated Cl⁻ secretion and transepithelial resistance. These effects were shown to require interaction of viable organisms with the apical surface of the monolayer and to be dependent upon organism number and duration of infection. Altered transport due to M. pulmonis was not merely a result of epithelial cell death as evidenced by the following: (i) active transport of Na⁺ and Cl⁻, albeit at reduced rates; (ii) normal cell morphology, including intact tight junctions, as demonstrated by electron microscopy; (iii) maintenance of a mean transepithelial resistance of 440 Ω/cm²; and (iv) lack of leakage of fluid from the basolateral to the apical surface of the monolayer. Alteration in epithelial ion transport in vitro is consistent with impaired pulmonary clearance and altered airway function in M. pulmonis-infected animals. Furthermore, the ability of M. pulmonis to alter transport without killing the host cell may explain its successful parasitism and long-term persistence in the host. Further study of the MTE-M. pulmonis model should elucidate the molecular mechanisms which mediate this reduction in transepithelial ion transport.Mycoplasmas, the smallest free-living prokaryotes, continue to be a significant cause of respiratory infections in a variety of animals, including humans (44). Their limited biosynthetic capability dictates that these organisms must both colonize and parasitize epithelial cell surfaces. It is therefore surprising that mycoplasmas produce diseases that are slowly progressing and chronic and yet often clinically inconspicuous. The molecular mechanisms responsible for this tenuous truce between the pathogen and the host cell have not yet been identified. Murine respiratory mycoplasmosis, a naturally occurring respiratory disease in laboratory rats and mice caused by Mycoplasma pulmonis (6, 8) and characterized by chronic, often lifelong tracheitis, bronchopneumonia, and bronchiectasis (7, 23, 28), would seem to be an ideal model with which to study these mechanisms. Although M. pulmonis-infected animals generate intense local and systemic immune responses (24, 33, 42, 43), they are incapable of eliminating the organism from the respiratory epithelium. Remodeling of the airways typically observed in infected animals, impairment of pulmonary clearance, and accumulation of mucus (23) suggest that M. pulmonis may compromise the ability of the epithelial cells to absorb and secrete fluid and electrolytes.Airway epithelial cells possess two major active transport processes, Na⁺ absorption and Cl⁻ section. Water, in turn, osmotically follows the transepithelial movement of these ions, thereby providing a fluid film between the mucus layer and the epithelial cell surface. The depth and composition of this fluid microenvironment must be carefully regulated to allow the exchange of gases and the humidification of the airway epithelium and to ensure proper mucociliary clearance (47, 49). The consequences of impaired fluid and electrolyte transport in the airways are strikingly apparent in cases of cystic fibrosis, a recessive genetic disease resulting from the loss of epithelial chloride channels (31, 48). The studies reported here were designed to determine whether M. pulmonis infection alters electrolyte transport across the murine tracheal epithelium.The effect of bacteria upon mammalian epithelia in the absence of immune and inflammatory cells can be systematically evaluated by using the Ussing chamber model. Short-circuit current (I_sc) studies have helped determine the mechanism by which cholera toxin (18) and other enterotoxins affect the intestinal mucosa (17, 35). Study of various types of epithelial cells by using the Ussing chamber model has also resulted in the identification of numerous microbial substances capable of directly altering the ion transport capacity of the airway epithelium (3, 20, 41).In the present study, control and M. pulmonis-infected mouse tracheal epithelial cells (MTEs) were evaluated in Ussing chambers. The results clearly show that M. pulmonis infection directly alters epithelial ion transport and that this alteration is species specific, dose and time related, and dependent upon the association of viable organisms with the apical cell surface.     

CD4+ T-cell memory: generation and multi-faceted roles for CD4+ T cells in protective immunity to influenza

Summary: We have outlined the carefully orchestrated process of CD4⁺ T‐cell differentiation from naïve to effector and from effector to memory cells with a focus on how these processes can be studied in vivo in responses to pathogen infection. We emphasize that the regulatory factors that determine the quality and quantity of the effector and memory cells generated include (i) the antigen dose during the initial T‐cell interaction with antigen‐presenting cells; (ii) the dose and duration of repeated interactions; and (iii) the milieu of inflammatory and growth cytokines that responding CD4⁺ T cells encounter. We suggest that heterogeneity in these regulatory factors leads to the generation of a spectrum of effectors with different functional attributes. Furthermore, we suggest that it is the presence of effectors at different stages along a pathway of progressive linear differentiation that leads to a related spectrum of memory cells. Our studies particularly highlight the multifaceted roles of CD4⁺ effector and memory T cells in protective responses to influenza infection and support the concept that efficient priming of CD4⁺ T cells that react to shared influenza proteins could contribute greatly to vaccine strategies for influenza.     

Rhabdoid tumor of the kidney with primitive neuroectodermal tumor of the central nervous system: Associated tumors with different histologic,cytogenetic, and molecular findings

Rhabdoid tumor of the kidney (RTK) is associated with tumors of the central nevous system (CNS) in approximately 15% of cases. We describe the clinical features, histologic and cytogenetic findings, and molecular analysis of renal and CNS tumors from the same patient. The histology of the renal tumor was consistent with rhabdoid tumor. The CNS tumor was a primitive neuroectodermal tumor (PNET). The karyotype of the RTK was normal male. The PNET of the brain demonstrated monosomy 22 as the only cytogenetic abnormality, similar to reported cases of malignant rhabdoid tumor of the brain, but dissimilar to nonrandom cytogenetic findings in other CNS PNETs. Molecular cytogenetic and DNA marker studies confirmed loss of chromosome 22 in this patient's brain tumor. DNA allelotyping showed retention of both parental chromosome 22 alleles in the RTK and loss of the maternal allele in the PNET. Evaluation of additional RTKs and brain tumors occurring in the same patient may provide insight into the origins and relationships of these enigmatic tumors.     

Persistent electrical coupling and locomotory dysfunction in the zebrafish mutant shocked

On initial formation of neuromuscular junctions, slow synaptic signals interact through an electrically coupled network of muscle cells. After the developmental onset of muscle excitability and the transition to fast synaptic responses, electrical coupling diminishes. No studies have revealed the functional importance of the electrical coupling or its precisely timed loss during development. In the mutant zebrafish shocked (sho) electrical coupling between fast muscle cells persists beyond the time that it would normally disappear in wild-type fish. Recordings from sho indicate that muscle depolarization in response to motor neuron stimulation remains slow due to the low-pass filter characteristics of the coupled network of muscle cells. Our findings suggest that the resultant prolonged muscle depolarizations contribute to the premature termination of swimming in sho and the delayed acquisition of the normally rapid touch-triggered movements. Thus the benefits of gap junctions during early synapse development likely become a liability if not inactivated by the time that muscle would normally achieve fast autonomous function.     

Monitoring CD4+ T cell responses against viral and tumor antigens using T cells as novel target APC

CD4+ T cells play an important role in the induction and maintenance of an effective antiviral and antitumor immune response. However, standardized monitoring of antigen-specific CD4+ T cells has not been established at the single-cell level. We now present a sensitive, specific, and simple methodology in which purified memory CD4+ T cells are expanded from PBMC in a single cycle of antigen-driven stimulation and quantitatively assayed by interferon-gamma ELISPOT. Issues of nonspecific background in assays were resolved with the use of innovative target cells, autologous PHA-expanded CD4+ T cells (T-APC). Remarkably, T-APC could not only present peptide epitopes from model antigens NY-ESO-1 and influenza nucleoprotein, but could also process full-length antigen endogenously expressed from recombinant fowlpox vector. This approach makes it possible to monitor CD4+ T cells in large series of patients, regardless of HLA haplotype, against the full peptide repertoire of a given antigen.