Sporophytic self-incompatibility genes and mating system variation in Arabis alpina.

BACKGROUND AND AIMS: Sporophytic self-incompatibility (SI) prevents inbreeding in many members of the Brassicaceae, and has been well documented in a variety of high-profile species. Arabis alpina is currently being developed as a model system for studying the ecological genetics of arctic-alpine environments, and is the focus of numerous studies on population structure and alpine phylogeography. Although it is highly inbreeding throughout most of its range, populations in central Italy have been identified that show inbreeding coefficients (F(IS)) more typical of self-incompatible relatives. The purpose of this study was to establish whether this variation is due to a functioning SI system. METHODS: Outcrossing rate estimates were calculated based on 16 allozyme loci and self-compatibility assessed based on controlled pollinations for six Italian populations that have previously been shown to vary in F(IS) values. Putative SRK alleles (the gene controlling the female component of SI in other Brassicaceae) amplified from A. alpina were compared with those published for other species. Linkage of putative SRK alleles and SI phenotypes was assessed using a diallel cross. KEY RESULTS: Functional avoidance of inbreeding is demonstrated in three populations of A. alpina, corresponding with previous F(IS) values. The presence is described of 15 putative SRK-like alleles, which show high sequence identity to known alleles from Brassica and Arabidopsis and the high levels of synonymous and nonsynonymous variation typical of genes under balancing selection. Also, orthologues of two other members of the S-receptor kinase gene family, Aly8 (ARK3) and Aly9 (AtS1) are identified. Further to this, co-segregation between some of the putative S-alleles and compatibility phenotypes was demonstrated using a full-sibling cross design. CONCLUSIONS: The results strongly suggest that, as with other species in the Brassicaceae, A. alpina has a sporophytic SI system but shows variation in the strength of SI within and between populations.

Population structure and historical biogeography of European Arabidopsis lyrata.

Understanding the natural history of model organisms is important for the effective use of their genomic resources. Arabidopsis lyrata has emerged as a useful plant for studying ecological and evolutionary genetics, based on its extensive natural variation, sequenced genome and close relationship to A. thaliana. We studied genetic diversity across the entire range of European Arabidopsis lyrata ssp. petraea, in order to explore how population history has influenced population structure. We sampled multiple populations from each region, using nuclear and chloroplast genome markers, and combined population genetic and phylogeographic approaches. Within-population diversity is substantial for nuclear allozyme markers (mean P=0.610, A(e)=1.580, H(e)=0.277) and significantly partitioned among populations (F(ST)=0.271). The Northern populations have modestly increased inbreeding (F(IS)=0.163 verses F(IS)=0.093), but retain comparable diversity to central European populations. Bottlenecks are common among central and northern Europe populations, indicating recent demographic history as a dominant factor in structuring the European diversity. Although the genetic structure was detected at all geographic scales, two clear differentiated units covering northern and central European areas (F(CT) =0.155) were identified by Bayesian analysis and supported by regional pairwise F(CT) calculations. A highly similar geographic pattern was observed from the distribution of chloroplast haplotypes, with the dominant northern haplotypes absent from central Europe. We conclude A. l. petraea's cold-tolerance and preference for disturbed habitats enabled glacial survival between the alpine and Nordic glaciers in central Europe and an additional cryptic refugium. While German populations are probable peri-glacial leftovers, Eastern Austrian populations have diversity patterns possibly compatible with longer-term survival.

Genetic diversity and phylogeography in two diploid ferns, Asplenium fontanum subsp. fontanum and A. petrarchae subsp. bivalens, in the western Mediterranean.

Asplenium fontanum subsp. fontanum and A. petrarchae subsp. bivalens are diploid rock ferns of limestone outcrops of the western Mediterranean region. Asplenium fontanum subsp. fontanum occurs from Valencia through northeastern Spain to the Alpes-Maritimes and Swiss Jura. Asplenium petrarchae subsp. bivalens occurs only on Majorca, in Valencia and possibly in southern Spain. We analysed allozyme and chloroplast genetic marker diversity in 75 populations of A. fontanum subsp. fontanum and 12 populations of A. petrarchae subsp. bivalens sampled from across their respective ranges. The two species show similar levels of species and population genetic diversity to one another and to other diploid European Asplenium taxa. Both are predominantly outbreeding, as indicated by F(IS) = 0.108 and 0.167 respectively. Substantial between-population differentiation results largely from differentiation between regions. Isolation by distance operates over limited geographic ranges, up to 50 km. In A. fontanum subsp. fontanum, the major geographical differentiation between Valencia and the rest of the taxon range probably represents an ancient range fragmentation. A less pronounced differentiation divides populations in the SW from those in the NE of the range, with evidence for a biogeographic link between the eastern Pyrenees and southeastern France. High diversity in the Pyrenees may either represent ancient population differentiation, or a suture zone. In A. petrarchae subsp. bivalens, populations on Majorca exhibit a subset of the genetic diversity present in Valencia, although the two regions are strongly differentiated by differing allele frequencies. Dispersal from the mainland may have founded Majorcan populations, although a role for in situ island survival cannot be excluded.

Genetic discontinuity, breeding-system change and population history of Arabis alpina in the Italian Peninsula and adjacent Alps.

Arabis alpina is a widespread plant of European arctic and alpine environments and belongs to the same family as Arabidopsis thaliana. It grows in all major mountain ranges within the Italian glacial refugia and populations were sampled over a 1300 km transect from Sicily to the Alps. Diversity was studied in nuclear and chloroplast genome markers, combining phylogeographical and population genetic approaches. Alpine populations had significantly lower levels of nuclear genetic variation compared to those in the Italian Peninsula, and this is associated with a pronounced change in within-population inbreeding. Alpine populations were significantly inbred (F(IS) = 0.553), possibly reflecting a change to the self-incompatibility system during leading edge colonization. The Italian Peninsula populations were approaching Hardy-Weinberg equilibrium (outbreeding, F(IS) = 0.076) and genetic variation was highly structured, consistent with independent local 'refugia within refugia' and the fragmentation of an established population by Quaternary climate oscillations. There is very little evidence of genetic exchange between the Alps and the Italian Peninsula main distribution ranges. The Alps functioned as a glacial sink for A. alpina, while the Italian Peninsula remains a distinct and separate long-term refugium. Comparative analysis indicated that inbreeding populations probably recolonized the Alps twice: (i) during a recent postglacial colonization of the western Alps from a Maritime Alps refugium; and (ii) separately into the central Alps from a source outside the sampling range. The pronounced geographical structure and inbreeding discontinuities are significant for the future development of A. alpina as a model species.

Espiroadenoma ecrino de presentación inusual en párpado / Eccrine spiradenoma of inusual presentation on the eyelid

Reportamos el caso de un espiroadenoma ecrino en una paciente de 90 años de edad, que empezó un año antes de la consulta con una lesión en el párpado. Se trata de un tumor benigno de la glándulas sudoríparas que es raro en el párpado. Al principio pensamos en otro tumor, como el carcinoma sebáceo, por su rápido crecimiento. La posibilidad de un tumor de glándulas sudoríparas debiera considerarse en el diagnóstico de tumores palpebrales.

We report a case of an eccrine spiradenoma in a 90 years old woman, who began a year before with the lesion on the eyelid. It is a begin sweat gland tumor, that is rare on the eyelid. At the beginning we thougth in an other tumor, like sebaceous carcinoma for its quick growth. The possibility of sweat gland tumor should be kept in mind in the diagnosis of eyelid tumours.

Recombination diversifies chloroplast trnF pseudogenes in Arabidopsis lyrata.

Extensive intraspecific variation in the chloroplast trnL(UAA)-trnF(GAA) spacer of model plant Arabidopsis lyrata is caused by multiple copies of a tandemly repeated trnF pseudogene undergoing parallel independent changes in copy number. Linkage disequilibrium and secondary structure analyses indicate that the diversification of pseudogene copies is driven by complex processes of structurally mediated illegitimate recombination. Disperse repeats sharing similar secondary structures interact, facilitating reciprocal exchange of structural motifs between copies via intramolecular and intermolecular recombinations, forming chimeric sequences and iterative expansion and contraction in pseudogene copy numbers. Widely held assumptions that chloroplast sequence evolution is simple and structural changes are informative are violated. Our findings have important implications for the use of this highly variable region in Brassicaceae studies. The reticulate evolution and nonindependent nucleotide substitution render the pseudogene inappropriate for standard phylogenetic reconstruction, but over short evolutionary timescales they may be useful for assessing gene flow, hybridization and introgression.

Ultraviolet light selection assay to optimize oligonucleotide correction of mutations in endogenous xeroderma pigmentosum genes.

Various oligonucleotide (ODN)-based approaches have been proposed for their ability to correct mutated genes at the normal chromosomal locations. However, the reported gene correction frequencies of these approaches have varied markedly in different experimental settings, including when different tissues or cell types are targeted. In order to find the optimal ODN-based approach for a specific target tissue, an assay system that allows direct comparison of the different methods on that tissue is necessary. Herein, we describe an XP-UVC selection assay that can be used to evaluate and compare gene correction frequencies in different cell types obtained from a xeroderma pigmentosum (XP) patient, following treatment by different ODN-based approaches. As an experimental example, the XP-UVC selection assay was used to assess the ability of chimeric RNA/DNA ODN to correct point mutations in the XPA gene. This assay can be used to assess and evaluate other types of ODN-based approaches, and to further optimize them.

Tumores estromales gastrointestinales / Stromal tumors gastrointestinal

Introducción: los tumores estromales gastrointestinales (GISTs)son un grupo eterogéneo de neoplasias mesenquimáticas, controvertidas para muchos en cuanto a morfología, criterios diagnósticos y comportamiento. Datos clínicos: se revisaron todos los tumores mesenquimáticos gastrointestinales de archivo del período 1992-2002. Se hallaron 7 casos de GISTs (25 por ciento de los tumores mesenquimáticos): 4 mujeres y 3 hombres, de 53 a 89 años; 5 fueron de estámago, 1 de intestino delgado y 1 de trascavidad de los epiplones. Métodos disgnósticos: el material se procesó según técnicas de rutina y se coloreó con H.E. Se efectuó IHQ para Actina Músculo Liso (AML), Desmina, S 100, CD 117. Hallazgo macroscópicos: todos los casos correspondieron a piezas quirúrgicas (gastrectomía, resección segmentaria intestinal o tumorectomía). El tamaño tumoral osciló entre 4 y 25 cm. Hallazgos microscópicos: consistieron en proliferaciones fusiformes, con grado variable de celularidad, atipía y actividad mitótica (AM)...

Tumores estromales gastrointestinales / Stromal tumors gastrointestinal

Introducción: los tumores estromales gastrointestinales (GISTs)son un grupo eterogéneo de neoplasias mesenquimáticas, controvertidas para muchos en cuanto a morfología, criterios diagnósticos y comportamiento. Datos clínicos: se revisaron todos los tumores mesenquimáticos gastrointestinales de archivo del período 1992-2002. Se hallaron 7 casos de GISTs (25 por ciento de los tumores mesenquimáticos): 4 mujeres y 3 hombres, de 53 a 89 años; 5 fueron de estámago, 1 de intestino delgado y 1 de trascavidad de los epiplones. Métodos disgnósticos: el material se procesó según técnicas de rutina y se coloreó con H.E. Se efectuó IHQ para Actina Músculo Liso (AML), Desmina, S 100, CD 117. Hallazgo macroscópicos: todos los casos correspondieron a piezas quirúrgicas (gastrectomía, resección segmentaria intestinal o tumorectomía). El tamaño tumoral osciló entre 4 y 25 cm. Hallazgos microscópicos: consistieron en proliferaciones fusiformes, con grado variable de celularidad, atipía y actividad mitótica (AM)...(AU)

Topical colchicine selection of keratinocytes transduced with the multidrug resistance gene (MDR1) can sustain and enhance transgene expression in vivo.

For skin gene therapy, achieving prolonged high-level gene expression in a significant percentage of keratinocytes (KC) is difficult because we cannot selectively target KC stem cells. We now demonstrate that topical colchicine treatment can be used to select, in vivo, KC progenitor cells transduced with the multidrug resistance gene (MDR1). When human skin equivalents containing MDR1-transduced KC were grafted onto immunocompromised mice, topical colchicine treatments significantly increased (7-fold) the percentage of KC expressing MDR1, compared to vehicle-treated controls, for up to 24 wk. Topical colchicine treatment also significantly enhanced the amount of MDR1 protein expressed in individual KC. Furthermore, quantitative real-time PCR analysis of MDR1 transgene copy number demonstrates that topical colchicine treatment selects and enriches for KC progenitor cells in the skin that contain and express MDR1. For clinical skin gene therapy applications, this in vivo selection approach promises to enhance both the duration and expression level of a desired therapeutic gene in KC, by linking its expression to the MDR1 selectable marker gene.

Polyploidy, phylogeography and Pleistocene refugia of the rockfern Asplenium ceterach: evidence from chloroplast DNA.

Chloroplast DNA sequences were obtained from 331 Asplenium ceterach plants representing 143 populations from throughout the range of the complex in Europe, plus outlying sites in North Africa and the near East. We identified nine distinct haplotypes from a 900 bp fragment of trnL-trnF gene. Tetraploid populations were encountered throughout Europe and further afield, whereas diploid populations were scarcer and predominated in the Pannonian-Balkan region. Hexaploids were encountered only in southern Mediterranean populations. Four haplotypes were found among diploid populations of the Pannonian-Balkans indicating that this region formed a northern Pleistocene refugium. A separate polyploid complex centred on Greece, comprises diploid, tetraploid and hexaploid populations with two endemic haplotypes and suggests long-term persistence of populations in the southern Mediterranean. Three chloroplast DNA (cpDNA) haplotypes were common among tetraploids in Spain and Italy, with diversity reducing northwards suggesting expansion from the south after the Pleistocene. Our cpDNA and ploidy data indicate at least six independent origins of polyploids.

Murine epidermal label-retaining cells isolated by flow cytometry do not express the stem cell markers CD34, Sca-1, or Flk-1.

Keratinocyte stem cells are present in the murine epidermis, based on both in vitro and in vivo evidence, and better characterization of these cells remains an active goal. Because keratinocyte stem cells are believed to cycle slowly, a good method for identification is based on their ability to retain nucleoside analog, such as bromodeoxyuridine. Adult stem cells have been identified in other tissues, including hematopoietic, neural, and skeletal muscle, and stem cell surface markers have been characterized. We wanted to determine if cell-surface markers present on both hematopoietic and skeletal muscle stem cells (CD34, Sca-1, and Flk-1) were also present on keratinocyte stem cells, and could be used to identify them. The cell-surface expression of cells that retained bromodeoxyuridine label for at least 21 d was compared with that of nonlabel-retaining cells. Double-labeling for flow cytometric analysis was employed, and label-retaining cells were found to lack expression of the tested markers. Beta1 integrin levels were also evaluated, and although high expression was found on label-retaining cells, it was not specific for these cells.

The hSkn-1a POU transcription factor enhances epidermal stratification by promoting keratinocyte proliferation.

Skn-1a is a POU transcription factor that is primarily expressed in the epidermis and is known to modulate the expression of several genes associated with keratinocyte differentiation. However, the formation of a stratified epidermis requires a carefully controlled balance between keratinocyte proliferation and differentiation, and a role for Skn-1a in this process has not been previously demonstrated. Here, our results show, surprisingly, that human Skn-1a contributes to epidermal stratification by primarily promoting keratinocyte proliferation and secondarily by enhancing the subsequent keratinocyte differentiation. In organotypic raft cultures of both primary human keratinocytes and immortalized HaCaT keratinocytes, human Skn-1a expression is associated with increased keratinocyte proliferation and re-epithelialization of the dermal substrates, resulting in increased numbers of keratinocytes available for the differentiation process. In these same raft cultures, human Skn-1a expression enhances the phenotypic changes of keratinocyte differentiation and the upregulated expression of keratinocyte differentiation genes. Conversely, expression of a dominant negative human Skn-1a transcription factor lacking the C-terminal transactivation domain blocks keratinocytes from proliferating and stratifying. Keratinocyte stratification is dependent on a precise balance between keratinocyte proliferation and differentiation, and our results suggest that human Skn-1a has an important role in maintaining epidermal homeostasis by promoting keratinocyte proliferation.

Advances in skin gene therapy.

Specific anatomical and biological properties make the skin a very interesting target organ for gene therapy approaches. Different cell types of the epidermis, such as keratinocytes, melanocytes, or dendritic cells, can be genetically modified to treat a broad spectrum of diseases, including genetically inherited skin disorders, tumour diseases, metabolic disorders and infectious diseases. The easy accessibility of skin suggests that different methods for gene delivery can be pursued, depending on the desired application. The approach used to deliver DNA to the skin will influence not only the efficiency of DNA delivery, but also the level and duration of transgene expression. Furthermore, the desired biological effect will also influence the decision of which gene transfer method is the best choice. Among the current challenges of cutaneous gene therapy are: optimising the efficiency of direct in vivo gene delivery; targeting specific epidermal cells, including keratinocyte stem cells; achieving sustained gene expression and regulating gene expression in vivo. This review summarises recent advances in the field of skin gene therapy and evaluates possible strategies to overcome obstacles and achieve successful clinical applications of skin gene therapy.

Nonviral skin gene therapy.

Nonviral skin gene therapy is an effective method to directly deliver and transiently express genes in the skin. Several different nonviral delivery methods have been successfully used and are analyzed here for their efficiency and efficacy in achieving specific therapeutic applications. For one important and frequently used application of nonviral skin gene therapy, genetic immunization, the types of resulting immune responses (Th1 versus Th2) will depend on which delivery method is used. In addition, we discuss the contributions of DNA as an immunostimulatory adjuvant in genetic immunization and how activation of skin dendritic cells and induction of IL-12 expression are mechanistically important in this process. Nonviral skin gene therapy has also been successfully used to enhance tumor regression in animal models, frequently by inducing a specific immune response against the tumor. In the future, nonviral skin gene therapy may be successfully used for the treatment of additional skin diseases if genes can be selectively delivered and expressed in specific skin cells, and if increased level and duration of gene expression can be achieved.

Selection of keratinocytes transduced with the multidrug resistance gene in an in vitro skin model presents a strategy for enhancing gene expression in vivo.

In gene therapy studies, achieving prolonged, high-level gene expression in a significant percentage of cells has been difficult. One solution to enhance expression would be to select for cells expressing both the desired gene and a linked selectable marker gene in a bicistronic vector. As a potential target tissue, the skin is easily accessible for safe topical application of a selecting agent that could lead to significant gene expression in a high percentage of keratinocytes. To test the feasibility of such an approach, a skin raft culture model was developed. Human keratinocytes were transduced with the multidrug resistance (MDR) gene, which confers resistance to a variety of cytostatic and antimitotic compounds, such as colchicine. While growing on acellular dermis, transduced keratinocytes were treated with various doses of colchicine (10-50 ng/ml). Colchicine treatment increased the percentage of keratinocytes expressing MDR to almost 100% in raft cultures, Significantly, keratinocytes in colchicine-treated, MDR-transduced raft cultures were able to proliferate normally and form a stratified, differentiated epidermis. This model suggests that topical selection for MDR-expressing keratinocytes in vivo should be feasible without hampering the biologic integrity of skin. Thus, topical selection leading to enhanced expression of a desired gene, linked to a resistance gene, holds future promise for skin gene therapy.

Characterization of the regulatory domains of the human skn-1a/Epoc-1/Oct-11 POU transcription factor.

The Skn-1a POU transcription factor is primarily expressed in keratinocytes of murine embryonic and adult epidermis. Although some POU factors expressed in a tissue-specific manner are important for normal differentiation, the biological function of Skn-1a remains unknown. Previous in vitro studies indicate that Skn-1a has the ability to transactivate markers of keratinocyte differentiation. In this study, we have characterized Skn-1a's transactivation domain(s) and engineered a dominant negative protein that lacked this transactivation domain. Deletional analysis of the human homologue of Skn-1a with three target promoters revealed the presence of two functional domains: a primary C-terminal transactivation domain and a combined N-terminal inhibitory domain and transactivation domain. Skn-1a lacking the C-terminal region completely lost transactivation ability, irrespective of the promoter tested, and was able to block transactivation by normal Skn-1a in competition assays. Compared with full-length, Skn-1a lacking the N-terminal region demonstrated either increased transactivation (bovine cytokeratin 6 promoter), comparable transactivation (human papillomavirus type 1a long control region), or loss of transactivation (human papillomavirus type 18 long control region). The identification of a primary C-terminal transactivation domain enabled us to generate a dominant negative Skn-1a factor, which will be useful in the quest for a better understanding of this keratinocyte-specific gene regulator.

A direct in vivo approach for skin gene therapy.

In vivo gene therapy is a direct and effective way to express genes in the epidermis. Plasmid DNA that contains the desired gene can be injected intradermally, and it is rapidly absorbed and expressed by the epidermis. Because gene expression following plasmid injection is transient, the two principal therapeutic uses of this approach are genetic immunization and the expression of biological response modifiers to treat skin disease.

Immunostimulatory oligodeoxynucleotides promote protective immunity and provide systemic therapy for leishmaniasis via IL-12- and IFN-gamma-dependent mechanisms.

Resistance to murine leishmaniasis correlates with development of a CD4(+) T helper 1 (Th1)-predominant immune response. To determine whether immunostimulatory CpG-containing oligodeoxynucleotides (CpG-ODN), known to promote a Th1 immune response, could provide protection from Leishmania infection, CpG-ODN and freeze-thawed (F/T) Leishmania major were coinjected intradermally into susceptible BALB/c mice. A Leishmania-specific Th1-predominant immune response was induced, and 40% of animals were protected from subsequent challenge with infectious organisms, with 0% protection of animals injected with F/T Leishmania organisms and PBS, F/T organisms and control ODN, or F/T organisms alone. More striking protection (65-95%) was seen in mice first infected with intact Leishmania organisms and then injected with CpG-ODN, either at the site of infection or at a remote site. To determine whether the therapeutic protection provided by CpG-ODN depended on IL-12 and IFN-gamma production, both IFN-gamma-deficient BALB/c mice and BALB/c mice treated with neutralizing anti-IL-12 mAb were first inoculated with Leishmania and then treated with either CpG-ODN, ODN, or PBS. None of these IFN-gamma-deficient mice survived (0/20, 0/20, and 0/20 respectively). Furthermore, neutralization of IL-12 completely abolished the therapeutic protection provided by CpG-ODN (0/20 mice surviving). We conclude that immunostimulatory DNA sequences likely exert systemic effects via IL-12 and IFN-gamma-dependent mechanisms and hold considerable promise as both vaccine adjuvants and potential therapeutic agents in the prevention and treatment of leishmaniasis.