Factors associated with different smoking status in European adolescents: results of the SEYLE study.

Early onset and long-term smoking are associated with physical and psychological health problems. The aim of the presented analysis was to investigate risk and influencing factors for different smoking status in a big sample of European adolescents. In the context of the "saving and empowering young lives in Europe" (SEYLE) study we surveyed 12,328 adolescents at the age of 13-17 from 11 countries. The survey took place in a school-based context using a questionnaire. Overall 58% reported the onset of ever-smoking under the age of 14 and 30.9% smoke on a daily basis. Multinomial logistic regression model showed significant positive associations between adolescent smoking and internalizing problems (suicidal behavior, direct self-injurious behavior, anxiety), externalizing problems (conduct problems, hyperactivity, substance consumption) and family problems (parental substance consumption, broken home). Our data show that smoking among adolescents is still a major public health problem and adolescents who smoke are at higher risk for mental problems. Further, adolescent smoking is associated with broken home families and parental behaviors. Therefore, early preventive measures are necessary not only for adolescents, but also for their parents.

[Complications after corneal cross-linking]. / Komplikationen nach Hornhautquervernetzung.

PURPOSE: The introduction of corneal cross-linking was associated with great expectations from patients as well as ophthalmologists. Previous results indicate that corneal cross-linking can slow down, stabilise or even reverse the progression of corneal ectasia in patients with keratoconus as well as in patients with ectasia after excimer laser ablations. We now describe our own experience with different protocols for corneal cross-linking and put these into perspective with the existing literature. METHODS: We undertook a retrospective analysis of all of our corneal cross-linking treatments from January 2007 to July 2014 using different protocols. In addition, we provide an overview of the literature regarding complications of corneal cross-linking. RESULTS: In our patient cohort we observed sterile infiltrates, transient cloudy opacifications, diffuse lamellar keratitis and a mycotic infection leading to corneal scarring. Moreover, even after transepithelial corneal cross-linking with different protocols, we have observed pain perception and epithelial disruption. At present there are few reports about infections or endothelial damage in the literature, however, induction of neoplasia is described. The incidence of complications appears to be low. CONCLUSION: Corneal cross-linking appears to have a high success rate and a low complication rate. Endothelial damage seems to be a far smaller problem than initially suspected. However, prospective studies with long-term follow-up are still lacking and it remains to be seen whether more cases of neoplasia following cross-linking will be reported. In our literature research we found no reports on re-treatments. Similarly, we found no study other than some case reports on keratoplasty after cross-linking. In view of rapidly changing treatment protocols further trials are warranted to monitor the benefits and complications of these modifications.

Automated recovery of the center of rotation in optical projection tomography in the presence of scattering.

Finding the center of rotation is an essential step for accurate three-dimensional reconstruction in optical projection tomography (OPT). Unfortunately current methods are not convenient since they require either prior scanning of a reference phantom, small structures of high intensity existing in the specimen, or active participation during the centering procedure. To solve these problems this paper proposes a fast and automatic center of rotation search method making use of parallel programming in graphics processing units (GPUs). Our method is based on a two step search approach making use only of those sections of the image with high signal to noise ratio. We have tested this method both in non-scattering ex vivo samples and in in vivo specimens with a considerable contribution of scattering such as Drosophila melanogaster pupae, recovering in all cases the center of rotation with a precision 1/4 pixel or less.

CDRAP is expressed in adult articular cartilage, but its expression is not significantly regulated in osteoarthritic chondrocytes.

OBJECTIVE: In this study we assessed the differential in vivo mRNA expression levels of CDRAP, a potential marker of cartilage degeneration. METHODS: Conventional and real time PCR in a large series of normal (n = 18) and late stage osteoarthritic (n = 24) cartilage specimens were performed. RESULTS: Conventional PCR analysis could demonstrate the presence of CDRAP mRNA in normal and osteoarthritic chondrocytes. Real time quantitative PCR confirmed the presence of CDRAP mRNA expression in normal articular chondrocytes in vivo (and in vitro). No significant up-regulation of CDRAP was observed in osteoarthritic chondrocytes in vivo. CONCLUSION: The presented results confirm expression of CDRAP by normal and osteoarthritic articular chondrocytes, but indicate that increased expression levels by chondrocytes are not the cause of the increased levels of CDRAP in the synovial fluid of patients with osteoarthritis.

MMP-9/gelatinase B is a gene product of human adult articular chondrocytes and increased in osteoarthritic cartilage.

OBJECTIVE: Collagen fibril degeneration involves initially the cleavage within the triple helix by the collagenases 1 (MMP-1) and 3 (MMP-13), but then mainly involves also the gelatinases A (MMP-2) and B (MMP-9). The objective of this study was to determine the quantitative expression levels as well as the distribution in normal and osteoarthritic cartilage of gelatinase B and in cultured articular chondrocytes with and without stimulation by Il-1Beta. METHODS: Conventional and real-time quantitative PCR technology and immunohistochemistry were used to determine gelatinase B expression on the mRNA and protein level. RESULTS: Conventional PCR analysis could demonstrate the presence of gelatinase B mRNA only in osteoarthritic chondrocytes. Real-time quantitative PCR confirmed the increased expression of gelatinase B mRNA expression in osteoarthritic chondrocytes. No significant up-regulation of gelatinase B was observed by Il-1Beta. Immunostaining for gelatinase B showed the presence of gelatinase B in a subset of normal and in a large portion of osteoarthritic chondrocytes with a more extended distribution in the latter. CONCLUSION: In osteoarthritic cartilage destruction, gelatinase B is involved in collagen destruction though still at a very much lower level than gelatinase A. Only a very small subset of normal adult articular chondrocytes express gelatinase B in vivo suggesting that gelatinase B unlike gelatinase A is hardly or only very focally involved in physiological collagen turnover.

Subtyping of osteoarthritic synoviopathy.

OBJECTIVE: Osteoarthritis research is traditionally concentrating on events within the degenerated articular cartilage. Changes in the synovial membrane are largely neglected. In fact, they are generally interpreted as secondary to the cartilage changes and not pathogenetically involved in the disease process. In this study, we present a systematic analysis of the synovial reaction pattern in early and late stages of the osteoarthritic disease process. METHODS: A large series of synovial specimens derived from early and late stage osteoarthritic cartilage disease were investigated by histological and immunohistochemical means for tissue architecture and inflammatory cell infiltrates. For comparison, also samples with rheumatoid arthritis, seronegative arthritis, and septic arthritis were included as well as normal synovial membrane specimens. RESULTS: In all specimens derived from patients with diagnosed osteoarthritis alterations of the synovial tissue were observed. A large spectrum of alterations was found in different stages of osteoarthritic joint disease and four different basic pattern of synovial reactions could be identified: (i) hyperplastic, (ii) inflammatory, (iii) fibrotic, and (iv) detritus-rich synoviopathy. CONCLUSION: We show that in all cases of clinically overt osteoarthritic joint disease significant synovial pathology is associated. Furthermore, our study clearly documents that in osteoarthritic synovium significant inflammation can occur. This is suggestive of a distinct pathogenetic role of the synovium also in osteoarthritic cartilage degeneration at least in a subset of cases.

Cell populations involved in pigmented villonodular synovitis of the knee.

OBJECTIVE: Pigmented villonodular synovitis (PVNS) of the knee is a tumor-like process of uncertain nature. We analyzed the involved cell populations, iron deposition, and cell proliferation in PVNS to propose a pathogenetic concept of this still elusive disease entity. METHODS: The study was performed on a series of 14 cases of localized PVNS of the knee. Histology and histochemistry were used to evaluate basic morphology and iron deposit distribution. Immunohistochemistry was performed to characterize the inflammatory cell infiltrate and to identify the proliferating cell compartments. In situ hybridization analysis using a cDNA probe against type I collagen was utilized to further characterize the mononuclear cell infiltrate. RESULTS: In addition to the classic features (mononuclear cell infiltrate, multinuclear giant cells, iron deposits, and stromal fibrosis) we observed a chronic inflammatory cell infiltrate in all PVNS samples, in which CD8 positive T cells were conspicuous. A high portion of non-phagocytotic cells resorbed iron and became CD68 positive. A proportion of mononuclear cells expressed type I collagen, thus resembling B synoviocytes. CONCLUSION: Our results suggest that preexisting chronic inflammation plays an important pathogenetic role in the PVNS disease process. Chronic inflammation increases the risk of articular bleeding and probably deranges the iron processing capacity of local synovial macrophages. The resulting iron overload could lead to a shift of iron storing cells from synovial macrophages to B synoviocytes and fibroblasts. A perpetuated proliferation and activation of these cells can explain why PVNS behaves like a neoplastic process.

Unusual 5' transcript complexity of plectin isoforms: novel tissue-specific exons modulate actin binding activity.

Plectin, the most versatile cytolinker identified to date, has essential functions in maintaining the mechanical integrity of skin, skeletal muscle and heart, as indicated by analyses of plectin-deficient mice and humans. Expression of plectin in a vast variety of tissues and cell types, combined with a large number of different binding partners identified at the molecular level, calls for complex mechanisms regulating gene transcription and expression of the protein. To investigate these mechanisms, we analyzed the transcript diversity and genomic organization of the murine plectin gene and found a remarkable complexity of its 5'-end structure. An unusually high number of 14 alternatively spliced exons, 11 of them directly splicing into plectin exon 2, were identified. Analysis of their tissue distribution revealed that expression of a few of them is restricted to tissues such as brain, or skeletal muscle and heart. In addition, we found two short exons tissue-specifically spliced into a highly conserved set of exons encoding the N-terminal actin binding domain (ABD), common to plectin and the superfamily of spectrin/dystrophin-type actin binding proteins. Using recombinant proteins we show that a novel ABD version contained in the muscle-specific isoform of plectin exhibits significantly higher actin binding activity than other splice forms. This fine tuning mechanism based on alternative splicing is likely to optimize the proposed biological role of plectin as a cytolinker opposing intense mechanical forces in tissues like striated muscle.

Is nitrocellulose filter binding really a universal assay for protein-DNA interactions?

The ability to bind to nitrocellulose is commonly accepted as being a universal property of proteins and has been widely used in many different fields of study. This property was first exploited in the study of DNA-binding proteins 30 years ago, in studies involving DNA binding by the lactose repressor (LacR) of Escherichia coli. Termed the filter-binding assay, it remains the quickest and easiest assay available for the study of protein-DNA interactions. However, the exact mechanism by which proteins bind to nitrocellulose remains uncertain. Given the supposedly universal nature of the interaction, we were surprised to notice that certain LacR variants were completely unable to bind simultaneously to DNA containing a single lac operator and nitrocellulose. Investigation of this loss of binding suggests that LacR requires a protein region that is both hydrophobic in nature and more or less unstructured, in order to bind to both nitrocellulose and DNA. In the case of wild-type, tetrameric LacR, the DNA-recognition domain that is not bound to DNA suffices. Dimeric LacR variants will only bind if they have certain C-terminal extensions. These experiments sound a cautionary note for the use of filter binding as an assay of choice, particularly in applications involving screening for the DNA-binding site of putative DNA-binding proteins.

Dimeric lac repressors exhibit phase-dependent co-operativity.

Transcription of the lac operon in Escherichia coli is repressed by the binding of Lac repressor (LacR) to lac operator O1, a pseudo-palindromic sequence centred 11 bp downstream of the transcription start. Full repression of the wild-type promoter by wild-type, tetrameric LacR requires the presence of at least two operator sequences that must not only be in close proximity to O1, 401 bp and 92 bp for the auxiliary operators O2 and O3, respectively, but must also be present on the same side of the DNA helix. LacR mutants lacking the C-terminal heptad repeat and thus only capable of dimer formation still repress, but at a much reduced level. Their repression of the lac promoter is comparable to repression by tetrameric LacR when both auxiliary operators are destroyed. We have examined the residual repression, by dimeric LacR, of a series of constructs containing a CAP-independent promoter and two lac operators, O1 and Oid, separated by a series of spacers increasing in size by single base-pair increments. Surprisingly, repression of these constructs still exhibits phase dependence. The periodicity of maxima is similar to the helical repeat of DNA in vivo, as measured by phase-dependent repression with tetrameric LacR, although the magnitude of repression is much smaller than that obtained in previous experiments with tetrameric LacR. Two additional variants of dimeric LacR with altered C termini that were tested also show phase dependence. Control experiments show that the presence of O1 is required for repression in this system. In the absence of O1, occupancy of the auxiliary operator does not lead to repression. The magnitudes of repression maxima correlate best with the overall basic nature of the C terminus. Weak, unspecific contacts by this region with DNA seem sufficient to explain the observed periodicity. It remains to be seen whether additional factors are also involved in this residual repression.

Operator search by mutant Lac repressors.

The Escherichia coli Lac and Gal repressors are two members of a large family of bacterial repressor proteins that share significant sequence and structural homology. Efficient repression by all family members requires specific binding to a site or sites close to the transcriptional start of the genes regulated. Both LacR and GalR have to bind to at least two sites for efficient repression, yet they differ in one important respect: LacR is a homotetramer whereas GalR is a homodimer. In an attempt to understand this difference, we studied the operator binding activity of a LacR variant that has the DNA-binding specificity of GalR (LacR-V17A18). A tetrameric version of this protein shows a 30-fold decrease in association rate to operator located on a long (lambda) DNA molecule, in comparison to wild-type LacR, while a dimeric version of this protein shows an unaltered association rate in comparison to dimeric LacR. This reduction in association rate correlates with a broadened DNA-binding specificity for base-pairs 4 and 5 of the operator: examination of an additional LacR variant with an even broader DNA-binding specificity indicates that a tetrameric version also shows a 30-fold decrease in association rate in comparison to wild-type LacR, while a dimeric version again shows an unaltered association rate in comparison to dimeric LacR. This difference in association rate in vitro correlates with whether a tetrameric or dimeric variant of LacR of a given DNA-binding specificity will repress lacZ under control of a single operator more efficiently in vivo. We therefore propose that the formation of stable homotetramers becomes a distinct disadvantage unless a high degree of DNA-binding specificity is also present, and demonstrate that this in indeed the case for GalR-mediated repression of the gal operon. This functional constraint seems to have influenced the evolution of the LacI-GalR family of repressors, most of which have a relatively broad specificity of DNA-binding and most of which form only stable homodimers.

[Iron deposits, cell populations and proliferative activity in pigmented villonodular synovitis of the knee joint]. / Eisenablagerungen, Zellpopulationen und proliferative Aktivität in der pigmentierten villonodulären Synovitis des Kniegelenks.

Pigmented villonodular synovitis (PVNS) of the knee is a tumor-like process of uncertain nature. A chronic inflammation as well as a neoplastic process have been proposed in the literature. The aim of our study was to characterise the prevalent inflammatory cells, the proliferating cell populations, and the iron deposit distribution in PVNS in order to get insights into pathogenetically relevant processes of this condition. Thirteen cases of PVNS of the knee as well as 8 normal controls were analysed histochemically for iron deposits and immunohistochemically for the distribution of vascular structures and inflammatory cell populations. Collagen type I expressing fibroblastic cells were identified by in situ hybridization. The proliferative cell compartment was characterized using MIB-1 staining. Our analysis showed no correlation between intra- or extracellular iron deposits and proliferation, giant cell formation, vascularity, number of CD 68-positive cells, and foam cell formation. Instead, iron deposits were associated with collagen matrix formation. All PVNS specimens showed a significant increase of chronic inflammatory infiltrates compared to all normal synovial membrane specimens investigated. The identification of the proliferative cell compartments showed that besides fibroblastic cells many of the mononuclear, partly CD 68 positive cells were Ki-67 positive. Foam cells, iron-loaded cells, and giant cells were, however, negative for the Ki-67 antigen. PVNS appears to originate from the interplay of proliferating, partly CD 68 positive mononuclear cells and fibroblasts, both activated by an excessive iron load. Giant cells probably develop by fusion of CD 68-positive histiocytic cells. Foam cells are most likely secondary to fatty tissue destruction.

Plectin transcript diversity: identification and tissue distribution of variants with distinct first coding exons and rodless isoforms.

Plectin is a widely expressed protein that is very large in size and that has all the attributes of a multifunctional crosslinking and organizing element of the cytoskeleton. It displays a multidomain structure, versatile binding activities, and subcellular localizations that enable it to strengthen cells against mechanical stress forces. Moreover, hereditary gene defects in plectin cause epidermolysis bullosa simplex (EBS)-MD, a severe skin blistering disease with muscular dystrophy. Here we report the analysis of the exonintron organization of the rat plectin gene and the identification of several different isoforms on the transcriptional level. We show that of 35 coding exons identified, 4 serve as alternative first exons splicing into the same successive exon 2, which is the first of 7 exons encoding a highly conserved actin-binding domain. RNase protection mapping of transcripts containing 3 of the identified 4 alternate first exons revealed their coexpression in rat glioma C6 cells and in a series of different rat tissues that we examined. Significant variations in expression levels of first exons indicated the possibility of tissue-specific promoter usage. In addition, plectin splice variants lacking exon 31 (> 3 kb), which encodes the entire rod domain of the molecule, were identified in a variety of rat tissues. This study provides first insights into a complex plectin gene regulatory machinery with similarities to that of dystrophin.

Repression of lac promoter as a function of distance, phase and quality of an auxiliary lac operator.

The tetrameric Lac repressor can bind simultaneously to two lac operators on the same DNA molecule, thereby including the formation of a DNA loop. We investigated the phasing dependence of DNA loop formation between lac operator O1 and an auxiliary ideal lac operator (O(id)) on the bacterial chromosome, with inter-operator distances varying from 57.5 to 1493.5 bp. Repression of a CAP-independent lac UV5 promoter by O1 at its natural position increased up to 50-fold in the presence of an optimally positioned auxiliary O(id)). Repression values alternated between local maxima and minima with a periodicity of 11.0 to 11.3 bp, suggesting that the chromosomal helical repeat is in this range in vivo. Repression increased significantly with decreasing inter-operator DNA length, indicating that the local Lac repressor concentration at O1 is crucial for tight repression. Maximal repression, attributed to stable DNA loop formation, was obtained at an operator spacing of 70.5 bp. Other repression maxima occurred at operator distances of 92.5 and 115.5 bp, corresponding to natural operator spacings in the lac and in the gal operon, respectively. Substitution of the auxiliary O(id) with the weaker binding lac operator O3 lowered repression efficiency, presumably due to the reduced local concentration of Lac repressor.

A basic tail increases repression by dimeric lac repressor.

Tetrameric Lac repressor achieves cooperative repression by binding simultaneously to O1 and to one of the auxiliary operators O2 or O3, thereby forcing the intervening DNA into a loop. Dimeric Lac repressor is not able to form DNA loops and consequently shows no cooperative repression. We constructed a dimeric Lac repressor mutant which exhibits increased repression to the lac operon that does not depend on specific operator-repressor-operator loops. This Lac repressor carries a synthetic tail of basic residues attached to its C terminus. With this construct, we observe an increase of the in vivo repression upon addition of auxiliary lac operators to a chromosomal lac operon controlled by O1. This suggests that the basic tail enables dimeric Lac repressor to enhance its repression by additional non-specific DNA contacts.

Quality and position of the three lac operators of E. coli define efficiency of repression.

Repression of the lac promoter may be achieved in two different ways: either by interference with the action of RNA polymerase or by interference with CAP activation. We investigated cooperative repression of the Escherichia coli lac operon by systematic conversion of its three natural operators (O1, O2 and O3) on the chromosome. We find that cooperative repression by tetrameric Lac repressor increases with both quality and proximity of the interacting operators. A short distance of 92 bp allows effective repression by two very weak operators (O3, O3). The cooperativity of lac operators is discussed in terms of a local increase of repressor concentration. This increase in concentration depends on flexible DNA which allows loop formation.

Genetic analysis of the leucine heptad repeats of Lac repressor: evidence for a 4-helical bundle.

Gel-filtration experiments indicate that a peptide (P2) composed of the basic region of GCN4 fused to the leucine heptad repeats of Lac repressor forms tetrameric aggregates. Gel-shift experiments were performed to determine the orientation of the helices in the tetrameric P2 aggregate. Sandwich-complex formation of peptide P2 with two DNA fragments containing two symmetrical CRE binding sites (5'-ATGACGTCAT-3') at a distance of 21 bp suggests antiparallel aggregation of the Lac leucine heptad repeats. Thus, we conclude that the leucine heptad repeats of Lac repressor have the ability to form homomeric 4-helical bundles with an antiparallel arrangement of the helices. This topology enables the two DNA fragments in the sandwich complexes to be held together by two tetramers of peptide P2. Replacement of the uncharged amino acids of the helical g and e positions of peptide P2 by the corresponding charged residues of GCN4 (peptide P4) results in a dimeric and parallel aggregation of the leucine heptad repeats, and consequently abolishes the potential to form sandwich structures. Similarly, a hybrid Lac repressor in which the GCN4 leucine zipper replaces the natural Lac leucine heptad repeats forms dimers only. It regains the ability to form tetramers when the charged amino acids in helical positions g and e are replaced by uncharged alanines.

Dimer-to-tetramer assembly of Lac repressor involves a leucine heptad repeat.

The C-terminus of Lac repressor is responsible for the formation of repressor tetramers from active dimers. If properly grafted, the C-terminus of Lac repressor (amino acids 331 to 360) converts Gal repressor dimers into tetramers. Amino acids 342 to 356 of Lac repressor contain a 4-3 hydrophobic repeat of four leucines and one valine. Systematic amino acid replacements of all residues in this region show that the protein-protein interaction between repressor dimers depends mainly on the hydrophobic residues of the 4-3 repeat, which is constitutive for coiled coils. Thus the tetramerization site of Lac repressor resembles the leucine zipper motif found in a family of eukaryotic transcription factors.

The three operators of the lac operon cooperate in repression.

We tested the effect of systematic destruction of all three lac operators of the chromosomal lac operon of Escherichia coli on repression by Lac repressor. Absence of just one 'pseudo-operator' O2 or O3 decreases repression by wild-type tetrameric Lac repressor approximately 2- to 3-fold; absence of both 'pseudo-operators' decreases repression greater than 50-fold. O1 alone represses under these conditions only approximately 20-fold. Dimeric active Lac repressor (iadi) represses the wild-type lac operon to about the same low extent. This indicates that cooperative interaction between lac operators is due to DNA loop formation mediated by tetrameric Lac repressor. Under conditions where loop formation is impossible, occupation of O3 but not of O2 may lead to weak repression. This suggests that under these conditions CAP activation may be inhibited and that stopping transcription at O2 does not significantly contribute to repression.

Recognition helices of lac and lambda repressor are oriented in opposite directions and recognize similar DNA sequences.

Exchanges in positions 1 and 2 of the putative recognition helix allow lac repressor to bind to ideal lac operator variants in which base pair 4 has been replaced. We show here that an Arg-22----Asn exchange in position 6 of the putative recognition helix of lac repressor abolishes lac repressor binding to ideal lac operator. This lac repressor variant, however, binds to a variant of the ideal lac operator 5' TTTGAGCGCTCAAA 3' in which the original G.C of position 6 has been replaced by T.A. This result and our previous data confirm our suggestion that the N terminus of the recognition helix of lac repressor enters the major groove close to the center of symmetry of lac operator and that its C terminus leaves the major groove further away from the center of symmetry. The consequences of this model are discussed in regard to various phage and bacterial repressor operator systems.