Cellular mechanisms of mutant connexins in skin disease and hearing loss

It has been demonstrated that distinct germline mutations within four connexin (Cx) genes, Cx26, Cx30, Cx31, and Cx30.3, underlie hearing loss and/or epidermal disease. Here, we describe two Cx26 mutations associated with skin disease. With the goal of understanding the mechanism(s) of Cx-associated human disease and how different mutations within the same Cx protein can result in different disorders, we performed a number of functional analyses investigating the cellular effects of disease-associated Cx mutations in keratinocytes and other cell types. Epidermal disease-associated proteins studied were primarily cytoplasmic with limited trafficking ability. FACS analysis of WT and mutant EGFP-Cx31 transfected keratinocytes revealed a high percentage of cell death associated with the skin disease-associated mutant Cx31 proteins.     

Functional studies of human skin disease- and deafness-associated connexin 30 mutations

Connexin 30 (Cx30) is a component of the gap junction complex. Dominant and recessive mutations in the GJB6 gene encoding Cx30 are associated with a variety of human inherited diseases primarily affecting the epidermis, hair, nail, and/or the inner ear. The underlying mechanism of disease associated with different GJB6 mutations such as the disruption of gap junction mediated intercellular communication is unknown. Towards understanding these disease mechanisms, transfection studies were performed in a keratinocyte cell line and in HeLa cells using EGFP tagged wildtype Cx30 and mutant Cx30 constructs harbouring dominant disease-associated GJB6 mutations. For all three of the skin disease-associated Cx30 mutations investigated, impaired trafficking of the protein to the plasma membrane was observed thus preventing the formation of functional Cx30 gap junctions. In contrast, the deafness-associated mutation T5M-Cx30/EGFP trafficked to the membrane but defective channel activity was observed following dye transfer studies.     

Localization of a gene (MCUL1) for multiple cutaneous leiomyomata and uterine fibroids to chromosome 1q42.3-q43

Dominant transmission of multiple uterine and cutaneous smooth-muscle tumors is seen in the disorder multiple leiomyomatosis (ML). We undertook a genomewide screen of 11 families segregating ML and found evidence for linkage to chromosome 1q42.3-q43 (maximum multipoint LOD score 5.40). Haplotype construction and analysis of recombinations permitted the minimal interval containing the locus, which we have designated "MCUL1," to be refined to an approximately 14-cM region flanked by markers D1S517 and D1S2842. Allelic-loss studies of tumors indicated that MCUL1 may act as a tumor suppressor. Identification of MCUL1 should have wide interest, since this gene may harbor low-penetrance variants predisposing to the common form of uterine fibroids and/or may undergo somatic mutation in sporadic leiomyomata.     

A case of spontaneous coronary artery disease regression

Coronary angiographic trials have demonstrated that lowering cholesterol can slow the progression of atherosclerosis, limit the formation of new lesions and enhance atherosclerotic regression together with reducing the incidence of clinical events (Waters D, 1996). Spontaneous regression of coronary atherosclerotic lesions is rare. We report the case of a patient with a severe within-stent restenotic lesion whose coronary disease spontaneously regressed 12 months after initial diagnosis, allowing for medical treatment of symptoms rather than repeated intervention. (Int J Cardiovasc Interventions 1999; 2: 121-123)     

Intermediate filament-membrane attachments function synergistically with actin-dependent contacts to regulate intercellular adhesive strength

By tethering intermediate filaments (IFs) to sites of intercellular adhesion, desmosomes facilitate formation of a supercellular scaffold that imparts mechanical strength to a tissue. However, the role IF-membrane attachments play in strengthening adhesion has not been directly examined. To address this question, we generated Tet-On A431 cells inducibly expressing a desmoplakin (DP) mutant lacking the rod and IF-binding domains (DPNTP). DPNTP localized to the plasma membrane and led to dissociation of IFs from the junctional plaque, without altering total or cell surface distribution of adherens junction or desmosomal proteins. However, a specific decrease in the detergent-insoluble pool of desmoglein suggested a reduced association with the IF cytoskeleton. DPNTP-expressing cell aggregates in suspension or substrate-released cell sheets readily dissociated when subjected to mechanical stress whereas controls remained largely intact. Dissociation occurred without lactate dehydrogenase release, suggesting that loss of tissue integrity was due to reduced adhesion rather than increased cytolysis. JD-1 cells from a patient with a DP COOH-terminal truncation were also more weakly adherent compared with normal keratinocytes. When used in combination with DPNTP, latrunculin A, which disassembles actin filaments and disrupts adherens junctions, led to dissociation up to an order of magnitude greater than either treatment alone. These data provide direct in vitro evidence that IF-membrane attachments regulate adhesive strength and suggest furthermore that actin- and IF-based junctions act synergistically to strengthen adhesion.     

Seq4SNPs: new software for retrieval of multiple, accurately annotated DNA sequences, ready formatted for SNP assay design

Background  

In moderate-throughput SNP genotyping there was a gap in the workflow, between choosing a set of SNPs and submitting their sequences to proprietary assay design software, which was not met by existing software. Retrieval and formatting of sequences flanking each SNP, prior to assay design, becomes rate-limiting for more than about ten SNPs, especially if annotated for repetitive regions and adjacent variations. We routinely process up to 50 SNPs at once.     

Mesozooplankton community structure changes in the Mfolozi–Msunduzi estuarine system,South Africa,during contrasting river flow conditions

The Mfolozi–Msunduzi estuarine system is subject to periodic dry and wet cycles, with subsequent changes in the abiotic and biotic characteristics of the system. The aim of the current study was to compare its mesozooplankton composition during relatively dry and wet periods. Mesozooplankton samples were collected between 2007 and 2010 in both the Mfolozi and the Msunduzi, covering a dry period between 2007 and 2008 and a period of relatively high freshwater inputs during 2009 and 2010. High flows during the wet period reduced the densities of most of the dominant estuarine mesozooplankton taxa in the Mfolozi Estuary, such as estuarine calanoids Pseudodiaptomus stuhlmanni (Poppe & Mrázek, 1895) and Acartiella natalensis (Connell & Grindley, 1974). The Msunduzi Estuary functioned as a reservoir from which recolonisation by estuarine taxa would quickly take place after the Mfolozi was scoured by floodwaters. Densities of dominant meroplankton taxa, such as zoeae of the crab Paratylodiplax blephariskios and Macrobrachium spp., were not noticeably different in the Mfolozi–Msunduzi system between the low- and high-flow periods.