Expression of the synthetic gene of an artificial DDT-binding polypeptide in Escherichia coli

This paper reports the expression of an artificial functional polypeptide in bacteria. The gene of a designed 24-residue DDT-binding polypeptide (DBP) was inserted between the BamHI and PstI cleavage sites of plasmid pUR291. The hybrid plasmid, pUR291-DBP, was cloned in Escherichia coli JM109. After induction by isopropyl-β-D-thiogalactopyranoside a fusion protein was expressed in which DBP was linked to the COOH-termiuus of β-galactosidase. DBP, which is stable to trypsin, was obtained by tryptic digestion of the fusion protein and subsequent fractionation of the tryptic peptides by reversed-phase h.p.l.c. Recombinant and chemically synthesized DBP showed identical chromatographic properties, amino acid composition, and chymotryptic digestion patterns. Both the β-galactosidase-DBP fusion and isolated recombinant DBP bound DDT. The fusion protein was 25 times as potent as the designed 24-residue DBP in activating a cytochrome P-450 model system using equimolar catalytic amounts of the two protein

Biosensors in Biomedical Research: Development and Applications of Gene Chips

Nucleic-acid hybridisation techniques are a central tool for the genetic analysis of biological systems. Gene chips are complex arrays of recombinant plasmids or oligonucleotides immobilised on a glass chip of only 1 cm2. This technology allows, for the first time, the multiparallel expression-analysis of thousands of genes. Gene chips will be indispensable tools for the upcoming analysis of the human genome, once the entire sequence is known

The voltage-dependent anion channel is the target for a new class of inhibitors of the mitochondrial permeability transition pore.

The relevance of the mitochondrial permeability transition pore (PTP) in Ca2+ homeostasis and cell death has gained wide attention. Yet, despite detailed functional characterization, the structure of this channel remains elusive. Here we report on a new class of inhibitors of the PTP and on the identification of their molecular target. The most potent among the compounds prepared, Ro 68-3400, inhibited PTP with a potency comparable to that of cyclosporin A. Since Ro 68-3400 has a reactive moiety capable of covalent modification of proteins, [3H]Ro 68-3400 was used as an affinity label for the identification of its protein target. In intact mitochondria isolated from rodent brain and liver and in SH-SY5Y human neuroblastoma cells, [3H]Ro 68-3400 predominantly labeled a protein of approximately 32 kDa. This protein was identified as the isoform 1 of the voltage-dependent anion channel (VDAC). Both functional and affinity labeling experiments indicated that VDAC might correspond to the site for the PTP inhibitor ubiquinone0, whereas other known PTP modulators acted at distinct sites. While Ro 68-3400 represents a new useful tool for the study of the structure and function of VDAC and the PTP, the results obtained provide direct evidence that VDAC1 is a component of this mitochondrial pore

Low rate of cardiac events in first-degree relatives of diagnosis-negative young sudden unexplained death syndrome victims during follow-up

BACKGROUND: Sudden unexplained death syndrome (SUDS) in young individuals often results from inherited cardiac disease. Accordingly, comprehensive examination in surviving first-degree relatives unmasks such disease in approximately 35% of the families. It is unknown whether individuals from diagnosis-negative families are at risk of developing manifest disease or cardiac events during follow-up.OBJECTIVE: This study aimed to study the prognosis of first-degree relatives of young SUDS victims, in whom the initial cardiologic and genetic examination did not lead to a diagnosis.METHODS: We retrieved vital status of surviving first-degree relatives from 83 diagnosis-negative families who presented to our cardiogenetics department between 1996 and 2009 because of SUDS in ≥1 relatives aged 1-50 years. Moreover, we contacted relatives who previously visited our center for detailed information.RESULTS: We obtained detailed information (median follow-up 6.6 years; interquartile range 4.7-9.6 years) in 340 of 417 first-degree relatives (81.5%) from 77 of 83 families (92.8%). Vital status, available in 405 relatives (97.1%), showed that 20 relatives (4.9%) died during follow-up, including 1 natural death before the age of 50. This girl belonged to a family with multiple cases of idiopathic ventricular fibrillation and SUDS, including another successfully resuscitated sibling during follow-up. Two hundred thirty-four of 340 first-degree relatives (68.8%) underwent cardiologic examination. Of these, 76 (32.5%) were reevaluated. Inherited cardiac disease was diagnosed in 3 families (3.6%).CONCLUSION: In first-degree relatives of young SUDS victims with no manifest abnormalities during the initial examination, the risk of developing manifest inherited cardiac disease or cardiac events during follow-up is low. This does not apply to families with obvious familial SUDS.</p

A New Yeast Poly(A) Polymerase Complex Involved in RNA Quality Control

Eukaryotic cells contain several unconventional poly(A) polymerases in addition to the canonical enzymes responsible for the synthesis of poly(A) tails of nuclear messenger RNA precursors. The yeast protein Trf4p has been implicated in a quality control pathway that leads to the polyadenylation and subsequent exosome-mediated degradation of hypomethylated initiator tRNA(Met) (tRNA(i) (Met)). Here we show that Trf4p is the catalytic subunit of a new poly(A) polymerase complex that contains Air1p or Air2p as potential RNA-binding subunits, as well as the putative RNA helicase Mtr4p. Comparison of native tRNA(i) (Met) with its in vitro transcribed unmodified counterpart revealed that the unmodified RNA was preferentially polyadenylated by affinity-purified Trf4 complex from yeast, as well as by complexes reconstituted from recombinant components. These results and additional experiments with other tRNA substrates suggested that the Trf4 complex can discriminate between native tRNAs and molecules that are incorrectly folded. Moreover, the polyadenylation activity of the Trf4 complex stimulated the degradation of unmodified tRNA(i) (Met) by nuclear exosome fractions in vitro. Degradation was most efficient when coupled to the polyadenylation activity of the Trf4 complex, indicating that the poly(A) tails serve as signals for the recruitment of the exosome. This polyadenylation-mediated RNA surveillance resembles the role of polyadenylation in bacterial RNA turnover

Prevalence of Age-Related Macular Degeneration in Europe: The Past and the Future

Purpose Age-related macular degeneration (AMD) is a frequent, complex disorder in elderly of European ancestry. Risk profiles and treatment options have changed considerably over the years, which may have affected disease prevalence and outcome. We determined the prevalence of early and late AMD in Europe from 1990 to 2013 using the European Eye Epidemiology (E3) consortium, and made projections for the future. Design Meta-analysis of prevalence data. Participants A total of 42 080 individuals 40 years of age and older participating in 14 population-based cohorts from 10 countries in Europe. Methods AMD was diagnosed based on fundus photographs using the Rotterdam Classification. Prevalence of early and late AMD was calculated using random-effects meta-analysis stratified for age, birth cohort, gender, geographic region, and time period of the study. Best-corrected visual acuity (BCVA) was compared between late AMD subtypes; geographic atrophy (GA) and choroidal neovascularization (CNV). Main Outcome Measures Prevalence of early and late AMD, BCVA, and number of AMD cases. Results Prevalence of early AMD increased from 3.5% (95% confidence interval [CI] 2.1%–5.0%) in those aged 55–59 years to 17.6% (95% CI 13.6%–21.5%) in those aged ≥85 years; for late AMD these figures were 0.1% (95% CI 0.04%–0.3%) and 9.8% (95% CI 6.3%–13.3%), respectively. We observed a decreasing prevalence of late AMD after 2006, which became most prominent after age 70. Prevalences were similar for gender across all age groups except for late AMD in the oldest age category, and a trend was found showing a higher prevalence of CNV in Northern Europe. After 2006, fewer eyes and fewer ≥80-year-old subjects with CNV were visually impaired (P = 0.016). Projections of AMD showed an almost doubling of affected persons despite a decreasing prevalence. By 2040, the number of individuals in Europe with early AMD will range between 14.9 and 21.5 million, and for late AMD between 3.9 and 4.8 million. Conclusion We observed a decreasing prevalence of AMD and an improvement in visual acuity in CNV occuring over the past 2 decades in Europe. Healthier lifestyles and implementation of anti–vascular endothelial growth factor treatment are the most likely explanations. Nevertheless, the numbers of affected subjects will increase considerably in the next 2 decades. AMD continues to remain a significant public health problem among Europeans

PLoS One

Age-related macular degeneration (AMD) is a common, progressive multifactorial vision-threatening disease and many genetic and environmental risk factors have been identified. The risk of AMD is influenced by lifestyle and diet, which may be reflected by an altered metabolic profile. Therefore, measurements of metabolites could identify biomarkers for AMD, and could aid in identifying high-risk individuals. Hypothesis-free technologies such as metabolomics have a great potential to uncover biomarkers or pathways that contribute to disease pathophysiology. To date, only a limited number of metabolomic studies have been performed in AMD. Here, we aim to contribute to the discovery of novel biomarkers and metabolic pathways for AMD using a targeted metabolomics approach of 188 metabolites. This study focuses on non-advanced AMD, since there is a need for biomarkers for the early stages of disease before severe visual loss has occurred. Targeted metabolomics was performed in 72 patients with early or intermediate AMD and 72 control individuals, and metabolites predictive for AMD were identified by a sparse partial least squares discriminant analysis. In our cohort, we identified four metabolite variables that were most predictive for early and intermediate stages of AMD. Increased glutamine and phosphatidylcholine diacyl C28:1 levels were detected in non-advanced AMD cases compared to controls, while the rate of glutaminolysis and the glutamine to glutamate ratio were reduced in non-advanced AMD. The association of glutamine with non-advanced AMD corroborates a recent report demonstrating an elevated glutamine level in early AMD using a different metabolomics technique. In conclusion, this study indicates that metabolomics is a suitable method for the discovery of biomarker candidates for AMD. In the future, larger metabolomics studies could add to the discovery of novel biomarkers in yet unknown AMD pathways and expand our insights in AMD pathophysiology