Two pregnancies after preimplantation genetic diagnosis for osteogenesis imperfecta type I and type IV

Osteogenesis imperfecta (OI) is an autosomal dominant genetic disorder characterized by the presence of brittle bones and decreased bone mass (osteopenia), as a result of mutations in the genes that encode the chains of type I collagen, the major protein of bone. The clinical features of the disease range from death in the perinatal period to normal life span with minimal increase in fractures. The present report describes two polymerase chain reaction (PCR)-based assays allowing preimplantation genetic diagnosis (PGD) on the one hand for OI type I, the mildest form, and on the other hand for OI type IV, which is intermediate in severity between OI type I and OI type III. In the couple referred for PGD for OI type I, the female partner carried a 1-bp deletion in exon 43 of the COL1A1 gene, resulting in a premature stop codon in exon 46. The synthesis of too little type I procollagen results from such a non-functional or COL1A1 null allele. In the other couple, referred for PGD for OI type IV, the male partner carried a G to A substitution in exon 19 of the COL1A2 gene, which results in an abnormal gene product due to an alphaGly247 (GGT) to Ser (AGT) substitution (G247S). Both mutations result in the loss of a specific restriction enzyme recognition site and can therefore be detected by PCR amplification followed by restriction fragment analysis. PCR amplification of genomic DNA of the parents-to-be with one of the two primers fluorescently labelled, followed by automated laser fluorescence (ALF) gel electrophoresis of the amplified and restricted fragments, allowed a distinction between the healthy and affected genotypes. PCR on single Epstein-Barr-virus (EBV)-transformed lymphoblasts resulted in acceptable amplification efficiencies (87% and 85% for OI type I and OI type IV respectively) and the allele drop-out (ADO) rate was assessed at 11.5% and 11.1% for OI type I and OI type IV respectively. With research blastomeres, 100% amplification rates were obtained and no contamination was observed in the blank controls, which validated the tests for clinical application. Embryos obtained after intracytoplasmic sperm injection (ICSI) were evaluated for the presence of the normal genotype of the non-affected parent. For OI type I, two frozen-thawed ICSI-PGD cycles and two fresh ICSI-PGD cycles were carried out for the same couple. The transfer of two unaffected embryos in the last cycle resulted in a twin pregnancy. A twin pregnancy was also achieved in one clinical ICSI-PGD cycle for OI type IV.     

A new Late-glacial and Holocene record of vegetation and fire history from Lago del Greppo, northern Apennines, Italy

Detailed Late-glacial and Holocene palaeoenvironmental records from the northern Apennines with a robust chronology are still rare, though the region has been regarded as a main area of potential refugia of important trees such as Picea abies and Abies alba. We present a new high-resolution pollen and stomata record from Lago del Greppo (1,442 m a.s.l., Pistoia, northern Apennines) that has been dated relying on 12 terrestrial plant macrofossils. Late-glacial woodlands became established before 13000 cal b.p. and were dominated by Pinus and Betula, although more thermophilous taxa such as Quercus, Tilia and Ulmus were already present in the Greppo area, probably at lower altitudes. Abies and Picea expanded locally at the onset of the Holocene at ca. 11500 cal b.p. Fagus sylvatica was the last important tree to expand at ca. 6500 cal b.p., following the decline of Abies. Human impact was generally low throughout the Holocene, and the local woods remained rather closed until the most recent time, ca. a.d. 1700–1800. The vegetational history of Lago del Greppo appears consistent with that of previous investigations in the study region. Late-glacial and Holocene vegetation dynamics in the northern Apennines are very similar to those in the Insubrian southern Alps bordering Switzerland and Italy, across the Po Plain. Similarities between the two areas include the Late-glacial presence of Abies alba, its strong dominance during the Holocene across different vegetation belts from the lowlands to high elevations, as well as its final fire and human-triggered reduction during the mid Holocene. Our new data suggest that isolated and minor Picea abies populations survived the Late-glacial in the foothills of the northern Apennines and that at the onset of the Holocene they moved upwards, reaching the site of Lago del Greppo. Today stands of Picea abies occur only in two small areas in the highest part of the northern Apennines, and they have become extinct elsewhere. Given the forecast global warming, these relict Picea abies stands of the northern Apennines, which have a history of at least 13,000 years, appear severely endangered.     

Chloroplast glutathione reductase: Purification and properties

Glutathione reductase was partially purified from isolated pea chloroplasts ( Pisum sativum L. cv. Progress #9). A 1600-fold purification was obtained and the purified enzyme had a specific activity of 26 μmol NADPH oxidized (mg protein)⁻¹ min⁻¹. The enzyme had a native molecular weight of approximately 156 kdalton and consisted of two each of two subunits of about 41 and 42 kdalton. The K_m for oxidized glutathione was 11 μ M and the K_m for NADPH was 1.7 μ M . Enzyme activity was affected by the ionic strength of the assay medium, and maximum activity was observed at an ionic strength of between 60 and 100 m M . The enzyme was inactivated by sulfhydryl modifying reagents and the presence of either oxidized glutathione or NADPH affected the extent of inactivation. Chloroplast glutathione reductase probably serves in the removal of photosynthetically derived H₂O₂ by reducing dehydroascorbate for ascorbate-linked reduction of H₂O₂. Intermediates of this reaction sequence, dehydroascorbate, ascorbate, reduced glutathione, and NADPH had no effect on enzymic activity.     

Using Sculptor and Situs for simultaneous assembly of atomic components into low-resolution shapes

We describe an integrated software system called Sculptor that combines visualization capabilities with molecular modeling algorithms for the analysis of multi-scale data sets. Sculptor features extensive special purpose visualization techniques that are based on modern GPU programming and are capable of representing complex molecular assemblies in real-time. The integration of graphics and modeling offers several advantages. The user interface not only eases the usually steep learning curve of pure algorithmic techniques, but it also permits instant analysis and post-processing of results, as well as the integration of results from external software. Here, we implemented an interactive peak-selection strategy that enables the user to explore a preliminary score landscape generated by the colors tool of Situs. The interactive placement of components, one at a time, is advantageous for low-resolution or ambiguously shaped maps, which are sometimes difficult to interpret by the fully automatic peak selection of colors. For the subsequent refinement of the preliminary models resulting from both interactive and automatic peak selection, we have implemented a novel simultaneous multi-body docking in Sculptor and Situs that softly enforces shape complementarities between components using the normalization of the cross-correlation coefficient. The proposed techniques are freely available in Situs version 2.6 and Sculptor version 2.0.     

Matrix-assisted laser desorption/ionization-tandem mass spectrometry with high resolution and sensitivity for identification and characterization of proteins

Although peptide mass fingerprinting is currently the method of choice to identify proteins, the number of proteins available in databases is increasing constantly, and hence, the advantage of having sequence data on a selected peptide, in order to increase the effectiveness of database searching, is more crucial. Until recently, the ability to identify proteins based on the peptide sequence was essentially limited to the use of electrospray ionization tandem mass spectrometry (MS) methods. The recent development of new instruments with matrix-assisted laser desorption/ionization (MALDI) sources and true tandem mass spectrometry (MS/MS) capabilities creates the capacity to obtain high quality tandem mass spectra of peptides. In this work, using the new high resolution tandem time of flight MALDI-(TOF/TOF) mass spectrometer from Applied Biosystems, examples of successful identification and characterization of bovine heart proteins (SWISS-PROT entries: P02192, Q9XSC6, P13620) separated by two-dimensional electrophoresis and blotted onto polyvinylidene difluoride membrane are described. Tryptic protein digests were analyzed by MALDI-TOF to identify peptide masses afterward used for MS/MS. Subsequent high energy MALDI-TOF/TOF collision-induced dissociation spectra were recorded on selected ions. All data, both MS and MS/MS, were recorded on the same instrument. Tandem mass spectra were submitted to database searching using MS-Tag or were manually de novo sequenced. An interesting modification of a tryptophan residue, a "double oxidation", came to light during these analyses.     

Stereoselective microbial dehalorespiration with vicinal dichlorinated alkanes

The suspected carcinogen 1,2-dichloroethane (1,2-DCA) is the most abundant chlorinated C(2) groundwater pollutant on earth. However, a reductive in situ detoxification technology for this compound does not exist. Although anaerobic dehalorespiring bacteria are known to catalyze several dechlorination steps in the reductive-degradation pathway of chlorinated ethenes and ethanes, no appropriate isolates that selectively and metabolically convert them into completely dechlorinated end products in defined growth media have been reported. Here we report on the isolation of Desulfitobacterium dichloroeliminans strain DCA1, a nutritionally defined anaerobic dehalorespiring bacterium that selectively converts 1,2-dichloroethane and all possible vicinal dichloropropanes and -butanes into completely dechlorinated end products. Menaquinone was identified as an essential cofactor for growth of strain DCA1 in pure culture. Strain DCA1 converts chiral chlorosubstrates, revealing the presence of a stereoselective dehalogenase that exclusively catalyzes an energy-conserving anti mechanistic dichloroelimination. Unlike any known dehalorespiring isolate, strain DCA1 does not carry out reductive hydrogenolysis reactions but rather exclusively dichloroeliminates its substrates. This unique dehalorespiratory biochemistry has shown promising application possibilities for bioremediation purposes and fine-chemical synthesis.     

Hydrophobic interaction between contiguous residues in the S6 transmembrane segment acts as a stimuli integration node in the BK channel

Large-conductance Ca²⁺- and voltage-activated K⁺ channel (BK) open probability is enhanced by depolarization, increasing Ca²⁺ concentration, or both. These stimuli activate modular voltage and Ca²⁺ sensors that are allosterically coupled to channel gating. Here, we report a point mutation of a phenylalanine (F380A) in the S6 transmembrane helix that, in the absence of internal Ca²⁺, profoundly hinders channel opening while showing only minor effects on the voltage sensor active–resting equilibrium. Interpretation of these results using an allosteric model suggests that the F380A mutation greatly increases the free energy difference between open and closed states and uncouples Ca²⁺ binding from voltage sensor activation and voltage sensor activation from channel opening. However, the presence of a bulky and more hydrophobic amino acid in the F380 position (F380W) increases the intrinsic open–closed equilibrium, weakening the coupling between both sensors with the pore domain. Based on these functional experiments and molecular dynamics simulations, we propose that F380 interacts with another S6 hydrophobic residue (L377) in contiguous subunits. This pair forms a hydrophobic ring important in determining the open–closed equilibrium and, like an integration node, participates in the communication between sensors and between the sensors and pore. Moreover, because of its effects on open probabilities, the F380A mutant can be used for detailed voltage sensor experiments in the presence of permeant cations.     

Elektronenmikroskopische Untersuchungen an der Interzellularsubstanz des menschlichen Knochengewebes

Zusammenfassung Die Interzellularsubstanz des Knochengewebes wurde im Durchstrahlungsbild elektronenmikroskopisch untersucht. Die aus der Licht-mikroskopie bekannten Knochenfibrillen setzen sich aus nur elektronenmikroskopisch sichtbaren Elementarfibrillen (Knochenfibrillen) und einer amorphen Kittsubstanz zusammen. In diese Kittsubstanz ist der Kalk eingelagert.Die Knochenfibrillen zeigen die charakteristische Querstreifung der Fibrillen aller Binde- und Stützgewebe. Bei der Bindegewebsversilberung nach Gömöri stimmt der Versilberungsmodus der Fibrillen des erwachsenen Knochens mit dem der reifen Fibrillen des Sehnenkollagens überein. Eine Differenzierung der Knochenfibrillen während der Entwicklung und Alterung läßt sich mit dieser Versilberungsmethode ebenfalls nachweisen. Es wurden Dickenunterschiede der Fibrillen im embryonalen Osteoid, im Faserknochen des Embryos und frühen Kindesalters und im lamellären Knochen festgestellt und tabellarisch zusammengefaßt. Auch die Periodenlängen der Fibrillen nehmen mit dem Alter des Knochengewebes zu. Zur Darstellung der Fibrillen wurden verschiedene Mazerations- und Fermentmethoden benutzt. Auch wurden mehrere Entkalkungsflüssigkeiten angewendet. Alle diese Methoden führen zu einer mehr oder weniger starken Quellung der Fibrillen. Als beste Methode zur Isolierung der Knochenfibrillen hat sich die Kombination von Trypsin- oder Papainverdauuung und Entkalkung mit Salpetersäure erwiesen. Die Knochenkittsubstanz wird mit zunehmendem Alter dichter und enthält sehr wenig Polysaccharide. Der Kalk ist in Form von ovalären und spindelförmigen Partikeln in die Kittsubstanz eingelagert. Die Größe der Kalkteilchen schwankt zwischen 15 und 130 m. Ihre Längsachse ist der Längsachse der Fibrillen parallel gerichtet. Die kleinsten Elemente liegen den Fibrillen, und zwar deren D-Teil an. Die Fibrillen selbst sind kalkfrei.Durchgeführt mit Unterstützung der Deutschen Forschungsgemeinschaft.     

Induction of auxin biosynthetic enzymes by jasmonic acid and in clubroot diseased Chinese cabbage plants

Nitrilase (NIT) and myrosinase are important enzymes for auxin biosynthesis in Brassicaceae, which is increased during clubroot disease. Therefore, NIT and myrosinase levels during club development and possible regulation mechanisms were investigated. In addition, the occurrence of different nitrilase isoforms in Chinese cabbage has been shown. Nitrilase activity was enhanced in infected roots during later stages of club development (35–42 days after inoculation). However, no differences in nitrilase mRNA levels between infected and healthy roots were found during symptom development. Myrosinase expression was increased in clubbed roots at slightly earlier time points (28 days after inoculation) and also at later time points during infection. The activities of tryptophan oxidizing enzyme (TrpOxE), which catalyzes the first step in tryptophan-dependent auxin biosynthesis in Brassicaceae, and nitrilase were enhanced after treatment with jasmonic acid (JA) and methyl jasmonate. Similarly, the amount of myrosinase mRNA was increased by JA. During clubroot disease the endogenous concentration of JA increased in infected roots 3–5 weeks after inoculation. From our results it can be concluded that: (1) de novo indole-3-acetic acid (IAA) biosynthesis plays a role for symptom development of clubroot disease in Brassicaceae during later developmental stages; and (2) JA which increased during club development, may be involved in the up-regulation of three enzymes important for IAA synthesis.