Induction of apoptosis by overexpression of the DNA-binding and DNA-PK-activating protein C1D

Apoptosis is induced in various tumor cell lines by vector-dependent overexpression of the conserved gene C1D that encodes a DNA-binding and DNA-PK-activating protein. C1D is physiologically expressed in 50 human tissues tested, which points to its basic cellular function. The expression of this gene must be tightly regulated because elevated levels of C1D protein, e.g. those induced by transient vector-dependent expression, result in apoptotic cell death. Cells transfected with C1D-expressing constructs show terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling of DNA ends. Transfections with constructs in which C1D is expressed in fusion with the (enhanced) green fluorescent protein from A. victoria (EGFP) allow the transfected cells to be identified and the morphological changes induced to be traced. Starting from intense nuclear spots, green fluorescence reflecting C1D expression increases dramatically at 12-24 hours post-transfection. Expression of C1D-EGFP protein is accompanied by morphological changes typical of apoptotic cell death, e.g. cytoplasmic vacuolation, membrane blebbing and nuclear disintegration. Cell shrinkage and detachment from extracellular matrix are observed in monolayer cultures while suspension cells become progressively flattened. The facility to differentiate between transfected and non-transfected cells reveals that non-transfected cells co-cultured with transfected cells also show the morphological changes of apoptosis, which points to a bystander effect. C1D-dependent apoptosis is not induced in cells with non-functional p53. Accordingly, C1D-induced apoptosis is discussed in relation to its potential to activate DNA-PK, which has been considered to act as an upstream activator of p53

Yeast Two-Hybrid: State of the Art

Genome projects are approaching completion and are saturating sequence databases. This paper discusses the role of the two-hybrid system as a generator of hypotheses. Apart from this rather exhaustive, financially and labour intensive procedure, more refined functional studies can be undertaken. Indeed, by making hybrids of two-hybrid systems, customised approaches can be developed in order to attack specific function-related problems. For example, one could set-up a "differential" screen by combining a forward and a reverse approach in a three-hybrid set-up. Another very interesting project is the use of peptide libraries in two-hybrid approaches. This could enable the identification of peptides with very high specificity comparable to "real" antibodies. With the technology available, the only limitation is imagination

Biofeedback for training balance and mobility tasks in older populations: a systematic review

<p>Abstract</p> <p>Context</p> <p>An effective application of biofeedback for interventions in older adults with balance and mobility disorders may be compromised due to co-morbidity.</p> <p>Objective</p> <p>To evaluate the feasibility and the effectiveness of biofeedback-based training of balance and/or mobility in older adults.</p> <p>Data Sources</p> <p>PubMed (1950-2009), EMBASE (1988-2009), Web of Science (1945-2009), the Cochrane Controlled Trials Register (1960-2009), CINAHL (1982-2009) and PsycINFO (1840-2009). The search strategy was composed of terms referring to biofeedback, balance or mobility, and older adults. Additional studies were identified by scanning reference lists.</p> <p>Study Selection</p> <p>For evaluating effectiveness, 2 reviewers independently screened papers and included controlled studies in older adults (i.e. mean age equal to or greater than 60 years) if they applied biofeedback during repeated practice sessions, and if they used at least one objective outcome measure of a balance or mobility task.</p> <p>Data Extraction</p> <p>Rating of study quality, with use of the Physiotherapy Evidence Database rating scale (PEDro scale), was performed independently by the 2 reviewers. Indications for (non)effectiveness were identified if 2 or more similar studies reported a (non)significant effect for the same type of outcome. Effect sizes were calculated.</p> <p>Results and Conclusions</p> <p>Although most available studies did not systematically evaluate feasibility aspects, reports of high participation rates, low drop-out rates, absence of adverse events and positive training experiences suggest that biofeedback methods can be applied in older adults. Effectiveness was evaluated based on 21 studies, mostly of moderate quality. An indication for effectiveness of visual feedback-based training of balance in (frail) older adults was identified for postural sway, weight-shifting and reaction time in standing, and for the Berg Balance Scale. Indications for added effectiveness of applying biofeedback during training of balance, gait, or sit-to-stand transfers in older patients post-stroke were identified for training-specific aspects. The same applies for auditory feedback-based training of gait in older patients with lower-limb surgery.</p> <p>Implications</p> <p>Further appropriate studies are needed in different populations of older adults to be able to make definitive statements regarding the (long-term) added effectiveness, particularly on measures of functioning.</p

Genome-wide search for breast cancer linkage in large Icelandic non-BRCA1/2 families

Abstract Introduction: A significant proportion of high-risk breast cancer families are not explained by mutations in known genes. Recent genome-wide searches (GWS) have not revealed any single major locus reminiscent of BRCA1 and BRCA2, indicating that still unidentified genes may explain relatively few families each or interact in a way obscure to linkage analyses. This has drawn attention to possible benefits of studying populations where genetic heterogeneity might be reduced. We thus performed a GWS for linkage on nine Icelandic multiple-case non-BRCA1/2 families of desirable size for mapping highly penetrant loci. To follow up suggestive loci, an additional 13 families from other Nordic countries were genotyped for selected markers. Methods: GWS was performed using 811 microsatellite markers providing about five centiMorgan (cM) resolution. Multipoint logarithm of odds (LOD) scores were calculated using parametric and nonparametric methods. For selected markers and cases, tumour tissue was compared to normal tissue to look for allelic loss indicative of a tumour suppressor gene. Results: The three highest signals were located at chromosomes 6q, 2p and 14q. One family contributed suggestive LOD scores (LOD 2.63 to 3.03, dominant model) at all these regions, without consistent evidence of a tumour suppressor gene. Haplotypes in nine affected family members mapped the loci to 2p23.2 to p21, 6q14.2 to q23.2 and 14q21.3 to q24.3. No evidence of a highly penetrant locus was found among the remaining families. The heterogeneity LOD (HLOD) at the 6q, 2p and 14q loci in all families was 3.27, 1.66 and 1.24, respectively. The subset of 13 Nordic families showed supportive HLODs at chromosome 6q (ranging from 0.34 to 1.37 by country subset). The 2p and 14q loci overlap with regions indicated by large families in previous GWS studies of breast cancer. Conclusions: Chromosomes 2p, 6q and 14q are candidate sites for genes contributing together to high breast cancer risk. A polygenic model is supported, suggesting the joint effect of genes in contributing to breast cancer risk to be rather common in non-BRCA1/2 families. For genetic counselling it would seem important to resolve the mode of genetic interaction

pWITCH: a versatile two-hybrid assay vector for the production of epitope/activation domain-tagged proteins both in vitro and in yeast.

We describe the construction of a new vector, pWITCH, designed to facilitate the characterisation of proteins encoded by novel cDNAs isolated using either a one- or two-hybrid assay. Expression of directionally cloned cDNAs is directed in vivo in Saccharomyces cerevisiae from the inducible GAL10 promoter and in vitro from the T7 promoter, while translation of the expressed cDNAs results in proteins which are tagged in vitro with a specific epitope and in vivo with both the epitope and the VP16 transcription activation domain. The principle of using multiple promoters each able to operate under different conditions to express different combinations of protein domains without the need for subcloning should be generally applicable

Melanocyte-specific gene expression: role of repression and identification of a melanocyte-specific factor, MSF.

For a gene to be transcribed in a tissue-specific fashion, expression must be achieved in the appropriate cell type and also be prevented in other tissues. As an approach to understanding the regulation of tissue-specific gene expression, we have analyzed the requirements for melanocyte-specific expression of the tyrosinase-related protein 1 (TRP-1) promoter. Positive regulation of TRP-1 expression is mediated by both an octamer-binding motif and an 11-bp element, termed the M box, which is conserved between the TRP-1 and other melanocyte-specific promoters. We show here that, consistent with its ability to activate transcription in a non-tissue-specific fashion, the M box binds the basic-helix-loop-helix factor USF in vitro. With the use of a combination of site-directed mutagenesis and chimeric promoter constructs, additional elements involved in regulating TRP-1 expression were identified. These include the TATA region, which appears to contribute to the melanocyte specificity of the TRP-1 promoter. Mutational analysis also identified two repressor elements, one at the start site, the other located at -240, which function both in melanoma and nonmelanoma cells. In addition, a melanocyte-specific factor, MSF, binds to sites which overlap both repressor elements, with substitution mutations demonstrating that binding by MSF is not required for repression. Although a functional role for MSF has not been unequivocally determined, the location of its binding sites leads us to speculate that it may act as a melanocyte-specific antirepressor during transcription of the endogenous TRP-1 gene

The effect of air pressure with cervical artificial insemination on the fertility of Awassi ewes synchronized with PGF2? [PGF2? ile senkronize edilen i·vesi koyunlari{dotless}nda hava basi{dotless}nci{dotless} ile servikal sun'i tohumlamani{dotless}n fertiliteye etkisi]

The aim of this study was to investigate the effect of air pressure on fertility during cervical artificial insemination with fresh semen in Awassi ewes. A total of 60 reproductively normal-cycling Awassi ewes (2-5 years old) were used in the experiment during the breeding season. All ewes were treated with the PG-7 days protocol consisting of two intramuscular doses of a PGF2? analogue administered 7 days apart. Animals were divided into two groups. The first group (n=30) was cervically inseminated (CAI). The second group (n=30) was cervically inseminated by means of an air pump (APCAI). Artificial inseminations were performed 48 h after the second PGF2? injection. Pregnancy was determined using ultrasonography on d 30 after AI. The pregnancy rate was found significantly higher in the APCAI group than in the CAI group (80.0% versus 46.7%) (P<0.01). The lambing rate was found to be greater in the group cervically inseminated with an air pump than in the group cervically inseminated with the conventional method (70.0 versus 46.7%). The insemination method used had a significant effect on lambing rate (P<0.05). Prolificacy was not affected by insemination methods (APCAI, 1.09; CAI, 1.14). It was concluded that cervical insemination with an air pump in Awassi ewes might improve clinical pregnancy rates by mechanisms yet to be defined

Positive and negative elements regulate a melanocyte-specific promoter.

Melanocytes are specialized cells residing in the hair follicles, the eye, and the basal layer of the human epidermis whose primary function is the production of the pigment melanin, giving rise to skin, hair, and eye color. Melanogenesis, a process unique to melanocytes that involves the processing of tyrosine by a number of melanocyte-specific enzymes, including tyrosinase and tyrosinase-related protein 1 (TRP-1), occurs only after differentiation from the melanocyte precursor, the melanoblast. In humans, melanogenesis is inducible by UV irradiation, with melanin being transferred from the melanocyte in the epidermis to the surrounding keratinocytes as protection from UV-induced damage. Excessive exposure to UV, however, is the primary cause of malignant melanoma, an increasingly common and highly aggressive disease. As an initial approach to understanding the regulation of melanocyte differentiation and melanocyte-specific transcription, we have isolated the gene encoding TRP-1 and examined the cis- and trans-acting factors required for cell-type-specific expression. We find that the TRP-1 promoter comprises both positive and negative regulatory elements which confer efficient expression in a TRP-1-expressing, pigmented melanoma cell line but not in NIH 3T3 or JEG3 cells and that a minimal promoter extending between -44 and +107 is sufficient for cell-type-specific expression. Assays for DNA-protein interactions coupled with extensive mutagenesis identified three factors, whose binding correlated with the function of two positive and one negative regulatory element. One of these factors, termed M-box-binding factor 1, binds to an 11-bp motif, the M box, which acts as a positive regulatory element both in TRP-1-expressing and -nonexpressing cell lines, despite being entirely conserved between the melanocyte-specific tyrosinase and TRP-1 promoters. The possible mechanisms underlying melanocyte-specific gene expression are discussed