Segregation of a mutation in CNGB1 encoding the β-subunit of the rod cGMP-gated channel in a family with autosomal recessive retinitis pigmentosa

Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous group of retinal diseases leading to blindness. By performing full genome linkage analysis in a consanguineous French family affected with severe autosomal recessive RP, we have excluded linkage to known loci involved in RP and mapped a novel locus to chromosome 16q13-q21 (Zmax=2.83 at theta=0 at the D16S3089 locus). Two candidate genes KIFC3 and CNGB1 mapping to this critical interval have been screened for mutations. The CNGB1 gene, which encodes the beta-subunit of the rod cGMP-gated channel, is mutated in the family presented in this study.     

Do proteins learn to evolve? The Hopfield network as a basis for the understanding of protein evolution

Correlations between amino-acid residues can be observed in sets of aligned protein sequences, and the analysis of their statistical and evolutionary significance and distribution has been thoroughly investigated. In this paper, we present a model based on such covariations in protein sequences in which the pairs of residues that have mutual influence combine to produce a system analogous to a Hopfield neural network. The emergent properties of such a network, such as soft failure and the connection between network architecture and stored memory, have close parallels in known proteins. This model suggests that an explanation for observed characters of proteins such as the diminution of function by substitutions distant from the active site, the existence of protein folds (superfolds) that can perform several functions based on one architecture, and structural and functional resilience to destabilizing substitutions might derive from their inherent network-like structure. This model may also provide a basis for mapping the relationship between structure, function and evolutionary history of a protein family, and thus be a powerful tool for rational engineering.     

Utilization of a multiple antigenic peptide as a calibration standard in the BAN50 single antibody sandwich ELISA for Aβ oligomers

Soluble amyloid-β (Aβ) oligomers are thought to be a cause of neurodegeneration and memory loss in Alzheimer disease (AD). We recently reported a newly developed enzyme linked immunosorbent assay (ELISA) for high molecular weight (HMW) Aβ oligomers in which the same Aβ monoclonal antibody, BAN50, was used for both capture and detection in a single antibody sandwich ELISA (SAS-ELISA) system. Our previous data suggest that this assay will be useful for the early diagnosis of AD, but its practical application to large-scale or longitudinal studies has been limited because of lack of a reliable calibration standard. In order to develop such a standard, we have now constructed a novel peptide using the multiple antigenic peptide (MAP) technique, where multiple epitopes of BAN50 were linked, via a spacer, to a branching lysine core. We show that the standard curve constructed from a 16-mer MAP covered the physiological range of signals obtained in the BAN50 SAS-ELISA from samples of human CSF, serum, and plasma. Furthermore, this 16-mer MAP is available in large quantities and is stable against freeze-thawing. We estimate that the signal per 1 pM of this standard corresponds to 1.54-5.0 pM of HMW Aβ oligomers. This MAP approach could also be used to provide an effective calibration standard for other SAS-ELISAs.     

On the Role of the SP1 Domain in HIV-1 Particle Assembly: a Molecular Switch?

Expression of a retroviral protein, Gag, in mammalian cells is sufficient for assembly of immature virus-like particles (VLPs). VLP assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We have investigated the role of SP1, a spacer between CA and NC in HIV-1 Gag, in VLP assembly. Mutational analysis showed that even subtle changes in the first 4 residues of SP1 destroy the ability of Gag to assemble correctly, frequently leading to formation of tubes or other misassembled structures rather than proper VLPs. We also studied the conformation of the CA-SP1 junction region in solution, using both molecular dynamics simulations and circular dichroism. Consonant with nuclear magnetic resonance (NMR) studies from other laboratories, we found that SP1 is nearly unstructured in aqueous solution but undergoes a concerted change to an α-helical conformation when the polarity of the environment is reduced by addition of dimethyl sulfoxide (DMSO), trifluoroethanol, or ethanol. Remarkably, such a coil-to-helix transition is also recapitulated in an aqueous medium at high peptide concentrations. The exquisite sensitivity of SP1 to mutational changes and its ability to undergo a concentration-dependent structural transition raise the possibility that SP1 could act as a molecular switch to prime HIV-1 Gag for VLP assembly. We suggest that changes in the local environment of SP1 when Gag oligomerizes on nucleic acid might trigger this switch.     

Electronic particle counting for evaluating the quality of air in operating theatres: a potential basis for standards?

Airborne particle counting in eight size ranges (0.5- greater than 20 microns), by computerized electronic equipment, was compared with the numbers of bacteria-carrying particles (BCP) assessed by slit sampling in ultra-clean and turbulently ventilated operating theatres. In the ultra-clean theatre the number of particles of 5-7 microns size range correlated with BCP while peaks in the numbers of particles less than 3 microns and greater than 15 microns corresponded with activity. Comparative relationships also occurred in the turbulently ventilated theatre but the use of this equipment in that environment cannot yet replace counts of airborne bacteria. We consider that electronic particle counting in the 0-20 microns size range may be used to judge the performance of a clean air operating theatre distribution system, including efficiency and integrity of the filter/seal systems and the presence or absence of entrainment of bacteria and other particles. The sampling techniques and analysis of particle concentration results described here may be a suitable basis for standards.     

Molecular links between X-inactivation and autosomal imprinting: X-inactivation as a driving force for the evolution of imprinting?

In classical Mendelian inheritance, each parent donates a set of chromosomes to its offspring so that maternally and paternally encoded information is expressed equally. The phenomena of X-chromosome inactivation (XCI) and autosomal imprinting in mammals violate this dogma of genetic equality. In XCI, one of the two female X chromosomes is silenced to equalize X-linked gene dosage between XX and XY individuals. In genomic imprinting, parental marks determine which of the embryo's two autosomal alleles will be expressed. Although XCI and imprinting appear distinct, molecular evidence now shows that they share a surprising number of features. Among them are cis-acting control centers, long-distance regulation and differential DNA methylation. Perhaps one of the most intriguing similarities between XCI and imprinting has been their association with noncoding and antisense RNAs. Very recent data also suggest the common involvement of histone modifications and chromatin-associated factors such as CTCF. Collectively, the evidence suggests that XCI and genomic imprinting may have a common origin. Here, I hypothesize that the need for X-linked dosage compensation was a major driving force in the evolution of genomic imprinting in mammals. I propose that imprinting was first fixed on the X chromosome for XCI and subsequently acquired by autosomes.     

The Duffy blood group is linked to the α-spectrin locus in a large pedigree with autosomal dominant inheritance of Charcot-Marie-Tooth disease type 1

Summary The -spectrin locus (SPTA) on chromsome 1 maps to 1q22–q25 and -spectrin specific probes detect restriction fragment length polymorphisms (RFLPs) with the endonucleases MspI and PvuII. The Duffy blood group (FY) has been mapped to the 1p21–q23 region. We found positive linkage between the -spectrin and the Duffy loci with a maximal Lod score of 3.81 at =0.0 using the computer program MLINK. This indicates that both loci are very closely linked and probably localized to 1q22–q23.     

δ1-Pyrroline-5-carboxylate reductase as a new target for therapeutics: inhibition of the enzyme from Streptococcus pyogenes and effects in vivo

Compounds able to interfere with amino acid biosynthesis have the potential to inhibit cell growth. In both prokaryotic and eukaryotic microorganisms, unless an ornithine cyclodeaminase is present, the activity of δ¹-pyrroline-5-carboxylate (P5C) reductase is mandatory to proline production, and the enzyme inhibition should result in amino acid starvation, blocking in turn protein synthesis. The ability of some substituted derivatives of aminomethylenebisphosphonic acid and its analogues to interfere with the activity of the enzyme from the human pathogen Streptococcus pyogenes was investigated. Several compounds were able to suppress activity in the micromolar range of concentrations, with a mechanism of uncompetitive type with respect to the substrate P5C and non-competitive with respect to the electron donor NAD(P)H. The actual occurrence of enzyme inhibition in vivo was supported by the effects of the most active derivatives upon bacterial growth and free amino acid content.     

F?rster-type energy transfer as a probe for changes in local fluctuations of the protein matrix

Much evidence, on both theoretical and experimental sides, indicates the importance of local fluctuations (in energy levels, conformational substates, etc.) of the macromolecular matrix in the biological activity of proteins. We describe here a novel application of the F?rster-type energy-transfer process capable of monitoring changes both in local fluctuations and in conformational states of macromolecules. A new energy-transfer parameter, f, is defined as an average transfer efficiency, [E], normalized by the actual average quantum efficiency of the donor fluorescence, [phi D]. A simple oscillator model (for a one donor-one acceptor system) is presented to show the sensitivity of this parameter to changes in amplitudes of local fluctuations. The different modes of averaging (static, dynamic, and intermediate cases) occurring for a given value of the average transfer rate, [kt], and the experimental requirements as well as limitations of the method are also discussed. The experimental tests were performed on the ribonuclease T1-pyridoxamine 5'-phosphate conjugate (a one donor-one acceptor system) by studying the change of the f parameter with temperature, an environmental parameter expectedly perturbing local fluctuations of proteins. The parameter f increased with increasing temperature as expected on the basis of the oscillator model, suggesting that it really reflects changes of fluctuation amplitudes (significant changes in the orientation factor, k2, as well as in the spectral properties of the fluorophores can be excluded by anisotropy measurements and spectral investigations). Possibilities of the general applicability of the method are also discussed.     

A frameshift mutation in exon 28 of the OPA1 gene explains the high prevalence of dominant optic atrophy in the Danish population: evidence for a founder effect

Dominant optic atrophy (DOA) is a hereditary optic neuropathy characterised by decreased visual acuity, colour vision deficits, centro-coecal scotoma and optic nerve pallor. The gene OPA1, encoding a dynamin-related GTPase, has recently been identified within the genetic linkage interval for the major locus for DOA on chromosome 3q28 and shown to harbour genetic aberrations segregating with disease in DOA families. The prevalence of the disorder in Denmark is reported to be the highest of any geographical location, suggestive of a founder effect. In order to establish the genetic basis of disease in a sample of 33 apparently unrelated Danish families, we screened DNA from affected members for OPA1 gene mutations by heteroduplex analysis and direct sequencing. A novel identical mutation in exon 28 (2826delT) was associated with DOA in 14 pedigrees and led to a frameshift and abnormal OPA1 protein -COOH terminus. Haplotype analysis of a region of approximately 1 Mb flanking the OPA1 gene using eight polymorphic markers revealed a common haplotype shared by all 14 patients; this haplotype was markedly over-represented compared with ethnically matched controls. Statistical analysis confirmed significant linkage disequilibrium with DOA over approximately 600 kb encompassing the disease mutation. We have therefore demonstrated that the relatively high frequency of DOA in Denmark is attributable to a founder mutation responsible for approximately 42% of the examined families and suggest that presymptomatic screening for the (2826delT) mutation may facilitate diagnosis and genetic counselling in a significant proportion of DOA patients of Danish ancestry.     

Induction of glutathione S-transferase in the freshwater bivalve Sphaerium corneum as a biomarker for short-term toxicity tests?

A study was carried out to examine the use of glutathione S-transferase (GST) in freshwater bivalves as a short-term biochemical marker for organic contaminants. In contrast with earlier reports, induction of GST activity towards 1-Chloro-2,4-dinitrobenzene (CDNB) was not found in any of the experiments performed. These included 10–12 days exposures to sediments spiked with 2,3′,4,4′,5-pentachlorobiphenyl, benzo[a]pyrene or lindane and 7–8 days exposures to dieldrin, lindane or benzo[a]pyrene in a water-only system. A nonsignificant decrease in GST activity (ca. − 20%) was found at the highest sediment concentration of lindane (10,000 μg/kg) and at the highest water concentrations of dieldrin (4.9 and 29 μg/l). Between experiments differences in GST activity were found that were ascribed to seasonal influences and maintenance in the laboratory. It is concluded that under the applied conditions glutathione S-transferase activity of Sphaerium corneum towards CDNB is not a suitable short-term biomarker with respect to the toxicants tested.     

Age as a Criterion for Setting Priorities in Health Care? A Survey of the German Public View

Although the German health care system has budget constraints similar to many other countries worldwide, a discussion on prioritization has not gained the attention of the public yet. To probe the acceptance of priority setting in medicine, a quantitative survey representative for the German public (n = 2031) was conducted. Here we focus on the results for age, a highly disputed criterion for prioritizing medical services. This criterion was investigated using different types of questionnaire items, from abstract age-related questions to health care scenarios, and discrete choice settings, all performed within the same sample. Several explanatory variables were included to account for differences in preference; in particular, interviewee''s own age but also his or her sex, socioeconomic status, and health status. There is little evidence that the German public accepts age as a criterion to prioritize health care services.     

Baculovirus immediate-early promoter-mediated expression of the Zeocin™ resistance gene for use as a dominant selectable marker in Dipteran and Lepidopteran insect cell lines

The antibiotic Zeocin, a derivative of phleomycin, was evaluated for use as a selection system in both dipteran and lepidopteran insect cell lines. Growth of Drosophila cell lines, Kc1 and SL2, was inhibited at Zeocin concentrations of 50 and 75 μg/ml, respectively, while the Spodoptera cell line, Sf9, was inhibited at a concentration of 250 μg/ml Zeocin. The mammalian cytomegalovirus (CMV) and Simian virus 40 (SV40) early promoters did not function in these insect cell lines. Several baculovirus-derived immediate-early (IE) promoters from the Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) and Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) were used to drive expression of the Zeocin resistance gene (ble) in these cell lines. The resulting plasmid vectors enabled selection of Zeocin-resistant cell lines within 3–4 weeks. Gene amplification events in the presence of increasing Zeocin concentrations were not detected using Southern blot analysis. Furthermore, the function of the baculovirus IE promoters, as demonstrated by β-galactosidase expression, was not detectable in a variety of mammalian cell lines tested. A cloning/shuttle vector, containing ten unique restriction sites, was constructed which allows for selection of Zeocin resistance in insect cell lines and in Escherichia coli.     

Is it possible to use the honey bee adult as a bioindicator for the detection of pesticide residues in plants?

Pesticide residues are usually determined by physical, chemical and biological methods. The simplicity and adaptability of bioassay methods have won their acceptance in the field of residue analysis. Theoretically, any organism that is susceptible to a pesticide may be used for its bioassay in any environmental sample. This means that such organism may serve as a bioindicator for the detection of certain pollutants. The susceptibility of honey bees (Apis melifera L.) to many insecticides commonly used in crop protection led to an attempt to use it as a bioindicator for the determination of residues of some insecticides in plant materials, as well as to detect toxicity hazards to honey bees of some commonly used insecticides. Results of this work which have been recently published may suggest "Yes" to answer the question posed in the title of this subject.