Cutaneous Mycobacterium chelonae infection in Edinburgh and the Lothians, South-East Scotland, U.K.

BACKGROUND: We reviewed all cases of Mycobacterium chelonae infection seen in our department between 1 January 2008 and 31 December 2012. OBJECTIVES: To review the epidemiology, clinical features and management of cutaneous M. chelonae in South-East Scotland, and to compare prevalence data with the rest of Scotland. METHODS: The Scottish Mycobacteria Reference Laboratory database was searched for all cases of cutaneous mycobacterial infections. RESULTS: One hundred and thirty-four cases of cutaneous mycobacterial infection were recorded. Sixty-three were tuberculous; of the remaining 71, M. chelonae was the most common nontuberculous organism (27 cases). National Health Service (NHS) Lothian Health Board was the area with highest incidence in the Scotland (12 cases). Three main groups of patients in the NHS Lothian Health Board contracted M. chelonae: immunosuppressed patients (n = 6); those who had undergone tattooing (n = 4); and others (n = 2). One case is, we believe, the first report of M. chelonae cutaneous infection associated with topical corticosteroid immunosuppression. The majority of patients were treated with clarithromycin monotherapy. CONCLUSION: The most prevalent nontuberculous cutaneous mycobacterial organism in Scotland is M. chelonae. The prevalence of M. chelonae in Edinburgh and the Lothians compared with the rest of Scotland is disproportionately high, possibly owing to increased local awareness and established facilities for mycobacterial studies. Immunosuppression with prednisolone appears to be a major risk factor. The first outbreak of tattoo-related M. chelonae infection in the U.K. has been reported. Clinicians should be aware of mycobacterial cutaneous infection and ensure that diagnostic skin samples are cultured at the optimal temperatures.

Mycobacterium chelonae infection: a complication of tattooing.

We describe an outbreak of Mycobacterium chelonae infection in four young immunocompetent patients who were tattooed by the same artist. All had been previously tattooed without complication, but following the latest tattooing session, they all developed a very similar papular eruption confined to skin that had been newly coloured light grey. On histological examination of the eruption, granulomatous inflammation with microabscess formation was seen, in association with the tattoo pigment. Skin cultures grown under optimal conditions grew M. chelonae, sensitive to clarithromycin, from one patient. M. chelonae was also cultured from the contents and nozzle of an opened bottle of light-grey ink from the tattoo parlour frequented by the patients. Dermatologists should consider mycobacterial infection in patients who develop inflammatory changes within a new tattoo.

Auger-mediated sticking of positrons to surfaces: evidence for a single-step transition from a scattering state to a surface image potential bound state.

We present the observation of an efficient mechanism for positron sticking to surfaces termed here Auger-mediated sticking. In this process the energy associated with the positrons transition from an unbound scattering state to a bound image potential state is coupled to a valence electron which can then have sufficient energy to leave the surface. Compelling evidence for this mechanism is found in a narrow secondary electron peak observed at incident positron kinetic energies well below the electron work function.

Ubinuclein, a novel nuclear protein interacting with cellular and viral transcription factors.

The major target tissues for Epstein-Barr virus (EBV) infection are B lymphocytes and epithelial cells of the oropharyngeal zone. The product of the EBV BZLF1 early gene, EB1, a member of the basic leucine-zipper family of transcription factors, interacts with both viral and cellular promoters and transcription factors, modulating the reactivation of latent EBV infection. Here, we characterize a novel cellular protein interacting with the basic domains of EB1 and c-Jun, and competing of their binding to the AP1 consensus site. The transcript is present in a wide variety of human adult, fetal, and tumor tissues, and the protein is detected in the nuclei throughout the human epidermis and as either grainy or punctuate nuclear staining in the cultured keratinocytes. The overexpression of tagged cDNA constructs in keratinocytes revealed that the NH(2) terminus is essential for the nuclear localization, while the central domain is responsible for the interaction with EB1 and for the phenotype of transfected keratinocytes similar to terminal differentiation. The gene was identified in tail-to-tail orientation with the periplakin gene (PPL) in human chromosome 16p13.3 and in a syntenic region in mouse chromosome 16. We designated this novel ubiquitously expressed nuclear protein as ubinuclein and the corresponding gene as UBN1.

Adherence to topical dermatological therapy: lessons from oral drug treatment.

Patients are remarkably nonadherent to medical treatment regimens across all diseases and classes of therapy, and it has been estimated that nonadherence to drug treatment is responsible for as many as 10% of all hospital admissions. Nonadherence to treatment also has significant negative effects on treatment outcomes across a wide range of diseases. Patient-related factors such as age, ethnicity, literacy (including health literacy), health beliefs, and socioeconomic conditions have been shown to influence adherence to oral therapy. Medication-related factors, such as regimen complexity and duration of treatment, also impact on adherence. Variables that significantly influence adherence to oral drugs have similar effects on adherence to topical therapy. Both educational and psychological interventions along with simplification of dosing regimens can significantly improve adherence to oral therapy and limited evidence indicates that these approaches are also effective in patients receiving topical therapy. There is very little information about the effects of dosing regimens on adherence to topical medical therapy. The advent of new drug formulations that permit once-daily or single-dose drug application will, however, permit evaluation of different topical treatment regimens on adherence and treatment outcomes in patients with dermatological disease.

The cellular oncogene c-myb can interact synergistically with the Epstein-Barr virus BZLF1 transactivator in lymphoid cells.

Regulation of replicative functions in the Epstein-Barr virus (EBV) genome is mediated through activation of a virally encoded transcription factor, Z (BZLF1). We have shown that the Z gene product, which binds to AP-1 sites as a homodimer and has sequence similarity to c-Fos, can efficiently activate the EBV early promoter, BMRF1, in certain cell types (i.e., HeLa cells) but not others (i.e., Jurkat cells). Here we demonstrate that the c-myb proto-oncogene product, which is itself a DNA-binding protein and transcriptional transactivator, can interact synergistically with Z in activating the BMRF1 promoter in Jurkat cells (a T-cell line) or Raji cells (an EBV-positive B-cell), whereas the c-myb gene product by itself has little effect. The simian virus 40 early promoter is also synergistically activated by the Z/c-myb combination. Synergistic transactivation of the BMRF1 promoter by the Z/c-myb combination appears to involve direct binding by the Z protein but not the c-myb protein. A 30-bp sequence in the BMRF1 promoter which contains a Z binding site (a consensus AP-1 site) is sufficient to transfer high-level lymphoid-specific responsiveness to the Z/c-myb combination to a heterologous promoter. That the c-myb oncogene product can interact synergistically with an EBV-encoded member of the leucine zipper protein family suggests c-myb is likely to engage in similar interactions with cellularly encoded transcription factors.

A methodological framework to assess the socio-economic impact of underground quarries: A case study from Belgian Limburg.

This study developed a methodology to assess the socio-economic impact of the presence and collapse of underground limestone quarries. For this we rely on case study evidence from Riemst, a village located in Eastern Belgium and use both secondary and primary data sources. A sinkhole inventory as well as data about the prevention costs provided by the municipality was used. To estimate the recreational values of the quarries, visitor data was obtained from the tourist office of Riemst. Next, two surveys were conducted among inhabitants and four real estate agents and one notary. The direct and indirect damages were assessed using respectively the repair cost and production and real estate value losses. The total yearly direct and indirect damage equals €415000 (±€85000) and more than half of it can be attributed to the depreciation of real estate (€230000). The quarries have recreational, cultural-historical and ecological values and thus generate societal benefits. The yearly recreational value was at least €613000 in 2012 values. The ecological and cultural-historical values augment to €180000 per year (in 2012 values). Further, our study indicates that the gains from filling up the quarries below the houses located above an underground limestone quarry outweigh the costs in the case study area. The net gain from filling up the underground quarry ranges €38700 to €101700 per house. This is only the lower bound of the net gain from filling up these underground quarries since preventive filling makes future collapses less likely so that future direct repair costs will be most likely smaller.

Chimeric c-Jun containing an heterologous homodimerization domain transforms primary chick embryo fibroblasts.

To investigate a possible role for c-Jun homodimers in c-Jun-mediated transformation, we designed two chimeric c-Jun derivatives, called c-Juneb1 and c-Jungcn4. In these chimeric derivatives the natural dimerization domain of c-Jun was replaced by the heterologous homodimerization domain of the Epstein-Barr virus EB1 or the yeast GCN4 transcription factor. Chick embryo fibroblasts chronically infected with retroviruses expressing c-Jun, c-Juneb1 or c-Jungcn4 are transformed. Infection with each construction results in sustained growth in low serum and development of colonies from single cells in agar with similar efficiencies. In contrast to c-Jun, c-Juneb1 and c-Jungcn4 confer additional phenotypic alterations related to in vitro transformation including a condensed cell morphology and ability to develop highly invasive, fast growing colonies in agar. These data suggest that c-Jun homodimers can transform chick embryo fibroblasts and activate cellular functions which influence cell morphology and invasive potential in agar. These findings are consistent with the notion that cellular transformation by c-jun is mediated by c-Jun homodimers.

The EBV early gene product EB2 transforms rodent cells through a signalling pathway involving c-Myc.

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus associated with several neoplasia. We present evidence here that the protein EB2, an EBV posttranscriptional activator, has transforming properties not only when expressed in established cell lines such as Rat1 or NIH3T3 but also in primary rat fibroblasts (REF). EB2 transformation in Rat1 cells correlates with an increase in the steady-state level of the cellular oncogenic protein c-Myc, and cotransfection of a plasmid expressing Max suppresses the transformation. These results implicate c-Myc in EB2-mediated cell transformation and help define the pathway by which this EBV early protein causes transformation.

Increased transforming activity of JunB and JunD by introduction of an heterologous homodimerization domain.

The closely-related proteins c-Jun, JunB and JunD form a family of transcription factors which require dimerization for DNA-binding and transcriptional activity. Dimerization is mediated by a conserved amphipathic alpha-helix located adjacent to a highly charged DNA-binding domain. The Jun proteins can form both homo- and heterodimers within the Jun family and can also cross-dimerize with the Fos proteins. When expressed at high levels in primary chicken cells, each mouse Jun displays distinct transforming capacities: c-Jun transforms efficiently, JunB transforms poorly, and JunD does not transform at all. The composition of the transforming dimers, however, is unknown. To study the activity of Jun-Jun homodimers we constructed artificial derivatives, denoted Juneb1, in which the naturally occurring dimerization domain has been replaced by an heterologous homodimerization domain from the Epstein-Barr virus transcription factor EB1. These derivatives were introduced into chicken cells and assayed for their ability to affect growth. Unexpectedly, all three Juneb1 proteins conferred a transformed phenotype to primary cultures, promoting sustained growth in low-serum medium and colony formation from single cells in agar. These data demonstrate that when forced to accumulate as homodimers, both JunB and JunD can transform cells. They also suggest that the poor transforming activity of JunB and the absence of transforming activity of JunD may be due to their inability to accumulate to high levels as homodimers.

Tumor induction by v-Jun homodimers in chickens.

To study the contribution of v-Jun homodimers to oncogenesis, we constructed artificial v-Jun derivatives in which the natural dimerization domain of v-Jun was replaced by an heterologous homodimerization domain from either the viral EB1 or the yeast GCN4 transcription factor. The resulting v-Jun chimeric proteins, called v-Juneb1 and v-Jungcn4, which can no longer dimerize with Jun or Fos, should only form homodimers in the cell. Helper-independent retroviruses expressing v-Jun, v-Juneb1 and v-Jungcn4 were generated. All three viruses transformed primary cultures of chick embryo cells with the same high efficiency and promoted local tumor growth after subcutaneous injection of infected cells in young animals. In contrast, after intravenous injection of viral suspensions into chick embryos, only the chimeric proteins produced internal tumors that were lethal. These tumors were leiomyosarcomas located within the liver and along the digestive tract. Thus, in vivo, v-Juneb1 and v-Jungcn4 are more potent oncoproteins than v-Jun. These data demonstrate that when forced to accumulate, v-Jun homodimers can induce tumors efficiently. They also show that the oncogenic potential of v-Jun can be regulated through the properties of its dimerization domain.

Molecular cloning and sequence analysis of the cDNA encoding rat liver cysteine sulfinate decarboxylase (CSD).

The taurine biosynthesis enzyme, cysteine sulfinate decarboxylase (CSD), was purified to homogeneity from rat liver. Three CSD peptides generated by tryptic cleavage were isolated and partially sequenced. Two of them showed a marked homology with glutamate decarboxylase and their respective position on the CSD amino acid sequence was postulated accordingly. Using appropriate degenerated primers derived from these two peptides, a PCR amplified DNA fragment was generated from liver poly(A)+ mRNA, cloned and used as a probe to screen a rat liver cDNA library. Three cDNAs, length around 1800 bp, were isolated which all contained an open reading frame (ORF) encoding a 493 amino acid protein with a calculated molecular mass of 55.2 kDa close to the experimental values for CSD. The encoded protein contained the sequence of the three peptides isolated from homogenous liver CSD. Our data confirm and significantly extend those recently published (Kaisaki et al. (1995) Biochim. Biophys. Acta 1262, 79-82). Indeed, an additional base pair found 1371 bp downstream from the initiation codon led to a shift in the open reading frame which extended the carboxy-terminal end by 15 amino acid residues and altogether modified 36 amino acids. The validity of this correction is supported by the finding that the corrected reading frame encoded a peptide issued from CSD tryptic cleavage that was not encoded anywhere in the CSD sequence previously reported.

A transcription enhancer acts in vitro over distances of hundreds of base-pairs on both circular and linear templates but not on chromatin-reconstituted DNA.

We have analyzed the effect of nucleosome formation and of the simian virus (SV40) enhancer on the efficiency of in vitro transcription. In a whole cell extract made from HeLa cells, nucleosome assembly on DNA results in the formation of chromatin-like complexes. However, transcription was detectable only when the DNA templates were partially or totally depleted of nucleosomes. On nucleosome-free templates, when the SV40 enhancer was present upstream from the complete SV40 early or rabbit beta-globin promoters, there was a five- to tenfold stimulation of specific transcription. When present upstream from its homologous promoter, the SV40 enhancer activated SV40 early transcription independently of its orientation with respect to the coding sequence. Point mutations known to impair the SV40 enhancer function in vivo had a similar effect in vitro. The extent of the enhancing effect was the same with linear or circular templates. When the SV40 enhancer was inserted upstream from the rabbit beta-globin gene, the activation of transcription was reduced with increasing distance between the enhancer and beta-globin upstream promoter elements, but was still significant over a distance of more than 400 base-pairs.

Human estrogen receptor (ER) gene promoter-P1: estradiol-independent activity and estradiol inducibility in ER+ and ER- cells.

Estrogen receptor (ER) is expressed at a low level in normal tissues such as breast and uterus but at a high level in breast and endometrial carcinomas. A proximal element (ERF-1) located between positions +133 and +204 relative to the promoter P1 major initiation site has been recently identified in ER+ cells and contributes to the differential promoter activity between ER+ and ER- cells. In this study, MCF7 and HeLa cells were transfected with chloramphenicol acetyltransferase constructs containing ER gene promoter P1 sequences. We show here that the sequences lying between nucleotides +13 to +212 are also essential for transcription at the ER gene promoter P1 in ER- cells, which do not express ERF-1. Interestingly, on gel shift experiments, a complex specific to ER- cells forms in the region spanning nucleotides +123 to +210. We also show that promoter P1 is responsive to estradiol in cells expressing endogenous (MCF7) or exogenous ER. We further demonstrate, using mutational analysis and gel retardation assays, that the three half-estrogen response elements located between nucleotides -420 and -892 are responsible for the estradiol inducibility of promoter P1. Because estradiol has a mitogenic effect on both breast and endometrial epithelial cells, our data would give an insight into the role of estrogens in the occurrence of breast and endometrial carcinomas.

Evidence for a functional glucocorticoid responsive element in the Epstein-Barr virus genome.

Glucocorticoids induce the expression of Epstein-Barr virus early antigens in latently infected Daudi cells. By sequence analysis, we found that fragment C of the BamHI digested Epstein-Barr virus B95-8 genome contains a region with a large degree of homology to the glucocorticoid responsive element of known glucocorticoid-regulated genes. By transfection experiments in Daudi and HeLa cells, different lengths of this region, cloned in front of the bacterial chloramphenicol acetyl transferase linked to the Herpes Simplex virus thymidine kinase promoter (pBLCAT.2), were assayed for their responsiveness to dexamethasone; our results led us to the conclusion that the hormonal effect observed was mediated by a minimal sequence of 15 base pairs presenting 85% homology with the consensus glucocorticoid responsive element sequence.

Kaposi's sarcoma-associated herpesvirus and Kaposi's sarcoma.

Kaposi's sarcoma-associated herpesvirus (KSHV) is present in all epidemiologic forms of Kaposi's sarcoma (KS). The KSHV genome contains several open reading frames which are potentially implicated in the development of KS. Some are unique to KSHV; others are homologous to cellular genes. The putative role of these genes in the genesis of KS is discussed.

Grafting peptides onto polystyrene microplates for ELISA.

Three peptides corresponding respectively to two Epstein-Barr viral epitopes and to the c-erbB-2 oncogene product were synthesized with the aim of developing an immunoenzymatic assay. Preliminary experiments indicated that the efficiency of the assay was profoundly affected by the nature of the solid phase for each peptide. In order to optimize the assay the three peptides were covalently coupled to functionalized polystyrene microplates which were used to immobilize both haptens and nucleic acids in a previous study. The results obtained indicate that the use of the carboxylated surfaces permits the linking strategy to be adapted to each peptide. Moreover, high sensitivities (5 x 10(-10)-1 x 10(-13) M) were obtained using amounts of the peptides much lower than those used in the standard system.

The acidic activation domain of the Epstein-Barr virus transcription factor R interacts in vitro with both TBP and TFIIB and is cell-specifically potentiated by a proline-rich region.

In cells latently infected with Epstein-Barr virus (EBV), the expression of two viral transactivators, EB1 and R, is responsible for the switch from latency to a productive cycle. R contains a DNA-binding/dimerization domain localized at the N-terminus. The domain required for transcriptional activation is localized at the C-terminus and contains two regions of very different amino acid composition. The first is very rich in prolines, whereas the second is rich in acidic residues and contains two potential alpha-helices. We investigated the activation potential of these subregions when linked to the heterologous Gal4 DNA-binding domain. We found that the acidic region--more precisely, the second putative alpha-helix--is an activating domain. In contrast, the proline-rich region is insufficient by itself for activation but collaborates with the acidic region in a cell-specific manner to make transactivation more efficient. We demonstrated that R interacts in vitro with the basal transcription factors TBP and TFIIB, and that the acidic domain of R mediates these interactions.