Band-specific localization of the microsatellite at D13S71 by microdissection and enzymatic amplification.

Microsatellite DNA consists of tandemly repeated simple DNA sequence motifs, the number of these repeats being polymorphic. These recently described polymorphisms are ubiquitously distributed throughout the human genome and are highly informative, making them ideal markers for linkage analysis. Physical localization of these microsatellites is an important prerequisite for aligning physical and genetic maps. We have physically mapped the microsatellite at D13S71, which has previously been assigned to chromosome 13. Band-specific mapping of D13S71 to the distal part of band 13q32, near 13q33, was achieved by microdissection of GTG-banded chromosomes and subsequent enzymatic amplification with a heminested PCR approach. Analysis of a panel of somatic cell hybrids confirmed this localization. The technique presented may also be useful in a variety of complex mapping situations and whenever the precise localization of very small (as small as 70 bp) DNA probes is necessary.     

Characterizing the normal proteome of human ciliary body

Background

The ciliary body is the circumferential muscular tissue located just behind the iris in the anterior chamber of the eye. It plays a pivotal role in the production of aqueous humor, maintenance of the lens zonules and accommodation by changing the shape of the crystalline lens. The ciliary body is the major target of drugs against glaucoma as its inhibition leads to a drop in intraocular pressure. A molecular study of the ciliary body could provide a better understanding about the pathophysiological processes that occur in glaucoma. Thus far, no large-scale proteomic investigation has been reported for the human ciliary body.

Results

In this study, we have carried out an in-depth LC-MS/MS-based proteomic analysis of normal human ciliary body and have identified 2,815 proteins. We identified a number of proteins that were previously not described in the ciliary body including importin 5 (IPO5), atlastin-2 (ATL2), B-cell receptor associated protein 29 (BCAP29), basigin (BSG), calpain-1 (CAPN1), copine 6 (CPNE6), fibulin 1 (FBLN1) and galectin 1 (LGALS1). We compared the plasma proteome with the ciliary body proteome and found that the large majority of proteins in the ciliary body were also detectable in the plasma while 896 proteins were unique to the ciliary body. We also classified proteins using pathway enrichment analysis and found most of proteins associated with ubiquitin pathway, EIF2 signaling, glycolysis and gluconeogenesis.

Conclusions

More than 95% of the identified proteins have not been previously described in the ciliary body proteome. This is the largest catalogue of proteins reported thus far in the ciliary body that should provide new insights into our understanding of the factors involved in maintaining the secretion of aqueous humor. The identification of these proteins will aid in understanding various eye diseases of the anterior segment such as glaucoma and presbyopia.     

Mal de Meleda (MDM) caused by mutations in the gene for SLURP-1 in patients from Germany,Turkey, Palestine,and the United Arab Emirates

Mal de Meleda (MDM) or keratosis palmoplantaris transgrediens of Siemens is an autosomal recessive skin disorder characterized by diffuse palmoplantar keratoderma (PPK) and transgressive keratosis with an onset in early infancy. There is no associated involvement of other organs; however, a spectrum of clinical presentations with optional and variable features has been described. Mutations in the ARS (component B)-81/s gene ( LY6LS) on chromosome 8q24-qter, which encodes SLURP-1, have recently been identified in patients with MDM. Here, we have analyzed four MDM families for mutations in SLURP-1. In a large Palestinian pedigree with multiple consanguinity, patients are homozygous for a new mutation that substitutes an arginine for a conserved glycine residue at position 86. A different mutation in Turkish patients results in the same amino acid exchange. Some remarkable similarities are seen in the clinical picture of patients from both families. Patients of an Emirati Bedouin family have a homozygous alteration of the translation initiation codon. In a German family with no known consanguinity, we have shown pseudodominant inheritance. Three affected children and their affected mother are homozygous for the missense mutation W15R. Our findings indicate that the MDM type of transgressive PPK is caused by SLURP-1 mutations in patients from various origins and demonstrate allelic heterogeneity for mutations in SLURP-1.     

Role of epidermis-type lipoxygenases for skin barrier function and adipocyte differentiation

12R-lipoxygenase (12R-LOX) and epidermis-type LOX-3 (eLOX-3) are novel members of the multigene family of mammalian LOX. A considerable gap exists between the identification of these enzymes and their biologic function. Here, we present evidence that 12R-LOX and eLOX-3, acting in sequence, and eLOX-3 in combination with another, not yet identified LOX are critically involved in terminal differentiation of keratinocytes and adipocytes, respectively. Mutational inactivation of 12R-LOX and/or eLOX-3 has been found to be associated with development of an inherited ichthyosiform skin disorder in humans and genetic ablation of 12R-LOX causes a severe impairment of the epidermal lipid barrier in mice leading to post-natal death of the animals. In preadipocytes, a LOX-dependent PPARgamma activating ligand is released into the cell supernatant early upon induction of differentiation and available evidence indicates that this ligand is an eLOX-3-derived product. In accordance with this data is the observation that forced expression of eLOX-3 enhances adipocyte differentiation.     

Characterization of dental pulp stem/stromal cells of Huntington monkey tooth germs

Background

Activation by extracellular ligands of G protein-coupled (GPCRs) and tyrosine kinase receptors (RTKs), results in the generation of second messengers that in turn control specific cell functions. Further, modulation/amplification or inhibition of the initial signalling events, depend on the recruitment onto the plasma membrane of soluble protein effectors. High throughput methodologies to monitor quantitatively second messenger production, have been developed over the last years and are largely used to screen chemical libraries for drug development. On the contrary, no such high throughput methods are yet available for the other aspect of GPCRs regulation, i.e. protein translocation to the plasma membrane, despite the enormous interest of this phenomenon for the modulation of receptor downstream functions. Indeed, to date, the experimental procedures available are either inadequate or complex and expensive.

Results

Here we describe the development of a novel conceptual approach to the study of cytosolic proteins translocation to the inner surface of the plasma membrane. The basis of the technique consists in: i) generating chimeras between the protein of interests and the calcium (Ca²⁺)-sensitive, luminescent photo-protein, aequorin and ii) taking advantage of the large Ca²⁺ concentration [Ca²⁺] difference between bulk cytosolic and the sub-plasma membrane rim.

Conclusion

This approach, that keeps unaffected the translocation properties of the signalling protein, can in principle be applied to any protein that, upon activation, moves from the cytosol to the plasma membrane. Thus, not only the modulation of GPCRs and RTKs can be investigated in this way, but that of all other proteins that can be recruited to the plasma membrane also independently of receptor activation. Moreover, its automated version, which can provide information about the kinetics and concentration-dependence of the process, is also applicable to high throughput screening of drugs affecting the translocation process.     

Spontaneous Vegetation on Overburden Piles in the Coal Basin of Santa Catarina,Brazil

The objective of this work was to select indigenous vegetal species for restoration programs aiming at the regeneration of ombrophilous dense forest. Thirty‐five spoil piles located in the county of Sideropolis, Santa Catarina, that received overburden disposal for 39 years (1950–1989) were selected for study because they exhibited remarkable spontaneous regrowth of trees compared to surrounding spoil piles. Floristic inventory covered the whole area of the 35 piles, whereas survey on phytosociology and natural regeneration studies were conducted in 70 plots distributed along the 35 piles. Floristic inventory recorded 83 species from 28 botanical families. Herbaceous terricolous plants constituted the predominant species (47.0%), followed by shrubs (26.5%), trees (19.3%), and vines (7.2%). Results from surveys on phytosociology and natural regeneration, focused on shrubs and trees, recorded incipient ecological succession. In addition, the most adapted species recorded on the overburden piles, as ranked by index of natural regeneration (RNT) plus importance value index (IVI), were as follows: Clethra scabra (RNT = 23.93%; IVI = 17.28%), Myrsine coriacea (RNT = 20.93%, IVI = 11.26%), Eupatorium intermedium (RNT = 7.56%, IVI = 0.40%), Miconia ligustroides (RNT = 5.84%, IVI = 2.37%), Ossaea amygdaloides (RNT = 3.84%, IVI = 1.30%), Tibouchina sellowiana (RNT = 3.29%, IVI = 1.94%), Eup. inulaefolium (RNT = 2.65%, IVI = 0.80%), and Baccharis dracunculifolia (RNT = 2.28%; IVI = 0.56%). High values of IVI and RNT exhibited by the exotic species Eucalyptus saligna (IVI = 21.73%, RNT = 51.41%) indicated strong competition between exotic and indigenous species. Severe chemical (acidic pH and lack of nutrients) and physical (coarse substrate and slope angle of 40–50°) characteristics displayed by the overburden piles constituted limitations to floristic diversity and size of indigenous trees, indicating the need for substrate reclamation prior to forest restoration.     

Krankheitsspezifische Hautmodelle

A variety of skin equivalent systems have been developed recently mainly for burn therapy but also for studies of the cell and molecular biology of dermatologic and immunologic disorders and for cosmetic and pharmaceutical research. Since European regulation forbids the use of animals to prove product safety in cosmetic products, several commercially available three-dimensional skin models were developed by the cosmetic and chemical industry and validated according to OECD and ECVAM regulations. Three-dimensional skin models consist of two compartments: one serves as a dermal equivalent, usually consisting of fibroblasts in type I collagen, onto which a terminally differentiating epidermis is placed. Up-to-date models are missing that mimic monogenic skin disorders or signs of disease in the skin caused by a systemic autoimmune disorder. We recently developed a three-dimensional skin model for congenital ichthyosis as an example for a keratinization disorder. The system is being validated and will be fundamental for studies of disturbed epidermal differentiation and pharmaceutical intervention.     

Familial Hemophagocytic Lymphohistiocytosis Type 5 (FHL-5) Is Caused by Mutations in Munc18-2 and Impaired Binding to Syntaxin 11

Rapid intracellular transport and secretion of cytotoxic granules through the immunological synapse requires a balanced interaction of several proteins. Disturbance of this highly regulated process underlies familial hemophagocytic lymphohistiocytosis (FHL), a genetically heterogeneous autosomal-recessive disorder characterized by a severe hyperinflammatory phenotype. Here, we have assigned FHL-5 to a 1 Mb region on chromosome 19p by using high-resolution SNP genotyping in eight unrelated FHL patients from consanguineous families. Subsequently, we found nine different mutations, either truncating or missense, in STXBP2 in twelve patients from Turkey, Saudi Arabia, and Central Europe. STXBP2 encodes syntaxin binding protein 2 (Munc18-2), involved in the regulation of vesicle transport to the plasma membrane. We have identified syntaxin 11, a SNARE protein mutated in FHL-4, as an interaction partner of STXBP2. This interaction is eliminated by the missense mutations found in our FHL-5 patients, which leads to a decreased stability of both proteins, as shown in patient lymphocytes. Activity of natural killer and cytotoxic T cells was markedly reduced or absent, as determined by CD107 degranulation. Our findings thus identify a key role for STXBP2 in lytic granule exocytosis.