Ultrafast Photoelimination of Nitrogen from Upper Excited States of Diazoalkanes and the Fate of Carbenes Formed in the Reaction.

The photochemical reactivity of diphenyldiazomethane 1 and phenyl 1- and 2-adamantyl diazomethanes 2 and 3, respectively, was investigated by transient absorption spectroscopy (TA). Photoelimination of N2 upon UV excitation takes place in the anti-Kasha ultrafast photochemical reaction from the upper excited singlet states to deliver singlet carbenes, which were, in the case of 1 and 2, detected by fs-TA. The reactivity of the carbenes differs with respect to the substituent at the carbene center. The singlet car-1 in a nonpolar solvent delivers the triplet carbene by intersystem crossing (ISC). Singlet car-2 does not undergo ISC but reacts in the intermolecular insertion reactions into C-H bonds. Car-3 has an α-C-H bond next to the carbene center and reacts rapidly in the intramolecular C-H insertion reaction to deliver alkene, precluding its detection by fs-TA. However, the isolation of ketone photoproducts from 3 is highly indicative of triplet car-3's intermediate formation. The TA spectra from the S1-S3 states of 1-3 were computed using time-dependent density functional theory, while the multiconfigurational perturbation theory to the second order was used for the absorption spectra of the corresponding singlet and triplet carbenes. The modeled and measured spectra are in good agreement, and the computations corroborate the assignments of the key short-lived intermediates.

Photoelimination of Nitrogen from Diazoalkanes: Involvement of Higher Excited Singlet States in the Carbene Formation.

Although diazoalkanes are important carbene precursors in organic synthesis, a comprehensive mechanism of photochemical formation of carbenes from diazoalkanes has not been proposed. Synergies of experiments and computations demonstrate the involvement of higher excited singlet states in the photochemistry of diazoalkanes. In all investigated diazoalkanes, excitation to S1 results in nonreactive internal conversion to S0. On the contrary, excitation to higher-lying singlet states (Sn, n > 1) drives the reaction toward a different segment of the S1/S0 conical intersection seam and results in nitrogen elimination and formation of carbenes.

ABCE1 Controls Ribosome Recycling by an Asymmetric Dynamic Conformational Equilibrium.

The twin-ATPase ABCE1 has a vital function in mRNA translation by recycling terminated or stalled ribosomes. As for other functionally distinct ATP-binding cassette (ABC) proteins, the mechanochemical coupling of ATP hydrolysis to conformational changes remains elusive. Here, we use an integrated biophysical approach allowing direct observation of conformational dynamics and ribosome association of ABCE1 at the single-molecule level. Our results from FRET experiments show that the current static two-state model of ABC proteins has to be expanded because the two ATP sites of ABCE1 are in dynamic equilibrium across three distinct conformational states: open, intermediate, and closed. The interaction of ABCE1 with ribosomes influences the conformational dynamics of both ATP sites asymmetrically and creates a complex network of conformational states. Our findings suggest a paradigm shift to redefine the understanding of the mechanochemical coupling in ABC proteins: from structure-based deterministic models to dynamic-based systems.

Structure of the ribosome post-recycling complex probed by chemical cross-linking and mass spectrometry.

Ribosome recycling orchestrated by the ATP binding cassette (ABC) protein ABCE1 can be considered as the final-or the first-step within the cyclic process of protein synthesis, connecting translation termination and mRNA surveillance with re-initiation. An ATP-dependent tweezer-like motion of the nucleotide-binding domains in ABCE1 transfers mechanical energy to the ribosome and tears the ribosome subunits apart. The post-recycling complex (PRC) then re-initiates mRNA translation. Here, we probed the so far unknown architecture of the 1-MDa PRC (40S/30S·ABCE1) by chemical cross-linking and mass spectrometry (XL-MS). Our study reveals ABCE1 bound to the translational factor-binding (GTPase) site with multiple cross-link contacts of the helix-loop-helix motif to the S24e ribosomal protein. Cross-linking of the FeS cluster domain to the ribosomal protein S12 substantiates an extreme lever-arm movement of the FeS cluster domain during ribosome recycling. We were thus able to reconstitute and structurally analyse a key complex in the translational cycle, resembling the link between translation initiation and ribosome recycling.

Monitoring of regional lung ventilation using electrical impedance tomography after cardiac surgery in infants and children.

Electrical impedance tomography (EIT) is a noninvasive method to monitor regional lung ventilation in infants and children without using radiation. The objective of this prospective study was to determine the value of EIT as an additional monitoring tool to assess regional lung ventilation after pediatric cardiac surgery for congenital heart disease in infants and children. EIT monitoring was performed in a prospective study comprising 30 pediatric patients who were mechanically ventilated after cardiac surgery. Data were analyzed off-line with respect to regional lung ventilation in different clinical situations. EIT data were correlated with respirator settings and arterial carbon dioxide (CO2) partial pressure in the blood. In 29 of 30 patients, regional ventilation of the lung could sufficiently and reliably be monitored by means of EIT. The effects of the transition from mechanical ventilation to spontaneous breathing after extubation on regional lung ventilation were studied. After extubation, a significant decrease of relative impedance changes was evident. In addition, a negative correlation of arterial CO2 partial pressure and relative impedance changes could be shown. EIT was sufficient to discriminate differences of regional lung ventilation in children and adolescents after cardiac surgery. EIT reliably provided additional information on regional lung ventilation in children after cardiac surgery. Neither chest tubes nor pacemaker wires nor the intensive care unit environment interfered with the application of EIT. EIT therefore may be used as an additional real-time monitoring tool in pediatric cardiac intensive care because it is noninvasive.

CHRNG genotype-phenotype correlations in the multiple pterygium syndromes.

BACKGROUND: Germline mutations in the CHRNG gene that encodes the γ subunit of the embryonal acetylcholine receptor may cause the non-lethal Escobar variant (EVMPS) or the lethal form (LMPS) of multiple pterygium syndrome (MPS). In addition CHRNG mutations and mutations in other components of the embryonal acetylcholine receptor may present with fetal akinesia deformation sequence (FADS) without pterygia. METHODS: In order to elucidate further the role of CHRNG mutations in MPS/FADS, this study evaluated the results of CHRNG mutation analysis in 100 families with a clinical diagnosis of MPS/FADS. RESULTS: CHRNG mutations were identified in 11/41 (27%) of families with EVMPS and 5/59 (8%) with LMPS/FADS. Most patients with a detectable CHRNG mutation (21 of 24 (87.5%)) had pterygia but no CHRNG mutations were detected in the presence of central nervous system anomalies. DISCUSSION: The mutation spectrum was similar in EVMPS and LMPS/FADS kindreds and EVMPS and LMPS phenotypes were observed in different families with the same CHRNG mutation. Despite this intrafamilial variability, it is estimated that there is a 95% chance that a subsequent sibling will have the same MPS phenotype (EVMPS or LMPS) as the proband (though concordance is less for more distant relatives). Based on these findings, a molecular genetic diagnostic pathway for the investigation of MPS/FADS is proposed.

Role of novel dimeric Photosystem II (PSII)-Psb27 protein complex in PSII repair.

The multisubunit membrane protein complex Photosystem II (PSII) catalyzes one of the key reactions in photosynthesis: the light-driven oxidation of water. Here, we focus on the role of the Psb27 assembly factor, which is involved in biogenesis and repair after light-induced damage of the complex. We show that Psb27 is essential for the survival of cyanobacterial cells grown under stress conditions. The combination of cold stress (30 °C) and high light stress (1000 µmol of photons × m(-2) × s(-1)) led to complete inhibition of growth in a Δpsb27 mutant strain of the thermophilic cyanobacterium Thermosynechococcus elongatus, whereas wild-type cells continued to grow. Moreover, Psb27-containing PSII complexes became the predominant PSII species in preparations from wild-type cells grown under cold stress. Two different PSII-Psb27 complexes were isolated and characterized in this study. The first complex represents the known monomeric PSII-Psb27 species, which is involved in the assembly of PSII. Additionally, a novel dimeric PSII-Psb27 complex could be allocated in the repair cycle, i.e. in processes after inactivation of PSII, by (15)N pulse-label experiments followed by mass spectrometry analysis. Comparison with the corresponding PSII species from Δpsb27 mutant cells showed that Psb27 prevented the release of manganese from the previously inactivated complex. These results indicate a more complex role of the Psb27 protein within the life cycle of PSII, especially under stress conditions.

Assembly of the water-oxidizing complex in photosystem II.

The efficient incorporation and assembly of calcium, chloride and manganese followed by photoactivation of the water-oxidizing complex (WOC) is a prerequisite for the unique water-splitting activity of photosystem II. This minireview summarizes the recent results on incorporation and storage of the inorganic cofactors, photoactivation of the WOC and assembly of the protein environment at the donor site of PSII in cyanobacteria with a special focus on the role of the Psb27 protein.

Osteopathia striata with cranial sclerosis owing to WTX gene defect.

Osteopathia striata with cranial sclerosis (OSCS) is an X-linked dominant condition marked by linear striations mainly affecting the metaphyseal region of the long bones and pelvis in combination with cranial sclerosis. Recently, the disease-causing gene was identified as the WTX gene (FAM123B), an inhibitor of WNT signaling. A correlation was suggested between the position of the mutation and male lethality. We performed genotype and phenotype studies using 18 patients from eight families with possible WTX gene defects and expanded the clinical spectrum of the affected females. All investigated families diagnosed with OSCS had WTX gene defects. One family had a WTX gene deletion; three of four point mutations were novel. The earlier reported WTX c.1072C>T was detected in four sporadic patients and appears to be a hotspot for mutations. Based on the nature of the mutation present in a surviving male patient, our data do not support the hypothesis raised by Jenkins et al. (2009) regarding a genotype-phenotype correlation for male lethality. The finding of a gene involved in WNT signaling as the cause of this sclerosing bone phenotype is not unexpected, but further functional studies are needed to explain the specific features. The WTX gene is mutated in different types of cancer, and it remains to be explained why osteopathia striata patients appear not to have an increased risk of cancer.

Caudal duplication syndrome with unilateral hypoplasia of the pelvis and lower limb and ventriculoseptal heart defect in a mother and features of VATER association in her child.

We report the case of a 22-year-old female with caudal duplication syndrome, who in addition to intestinal duplication, imperforate anus, a dydelphic uterus and a single kidney also had a ventricular septal defect and hypoplasia of the left pelvis, leg, labia majora and left side of a duplicated vagina. She gave birth to a male baby with features of the VATER association including a tracheooesophageal fistula, a ventriculoseptal defect, an atrial septal defect and mild hypospadias. We suggest that caudal duplication syndrome and the VATER association may overlap and our two cases suggest possible autosomal dominant inheritance.

Constitutional trisomy 8 and Behçet syndrome.

The characteristic clinical features of constitutional trisomy 8 include varying degrees of developmental delay, joint contractures and deep palmar and plantar creases. There is an established literature, which describes features of Behçet syndrome occurring in phenotypically normal individuals with myelodysplastic syndromes and trisomy 8 in their bone marrow. In this article, we describe four patients with constitutional trisomy 8, all with varying clinical phenotypes, who developed features of Behçet, in particular but not exclusively mucocutaneous ulceration. In addition, we examined gene copy numbers of the variable-number neutrophil defensin genes DEFA1A3 in one of the cases (case 1) and her parents, together with 14 cases of Behçet syndrome in comparison with 121 normal controls. The gene copy number was highest in case 1 (copy number 14) and was also increased in her parents (both copy number 9). However the mean copy number for DEFA1A3 among the 14 Behçet syndrome patients was actually lower (5.1) than among the controls (mean of 6.8 copies). Thus, we conclude that patients with constitutional trisomy 8 and those with trisomy 8 confined to the bone marrow are both at increased risk of developing features of Behçet syndrome. The mechanism may relate to increased chromosome 8 gene dosage with further analysis of candidate genes on chromosome 8 required.

Mutations in the pericentrin (PCNT) gene cause primordial dwarfism.

Fundamental processes influencing human growth can be revealed by studying extreme short stature. Using genetic linkage analysis, we find that biallelic loss-of-function mutations in the centrosomal pericentrin (PCNT) gene on chromosome 21q22.3 cause microcephalic osteodysplastic primordial dwarfism type II (MOPD II) in 25 patients. Adults with this rare inherited condition have an average height of 100 centimeters and a brain size comparable to that of a 3-month-old baby, but are of near-normal intelligence. Absence of PCNT results in disorganized mitotic spindles and missegregation of chromosomes. Mutations in related genes are known to cause primary microcephaly (MCPH1, CDK5RAP2, ASPM, and CENPJ).

Brief report: A case of autism associated with del(2)(q32.1q32.2) or (q32.2q32.3).

Autism is a neurodevelopmental disorder presenting in the first 3 years of life. Deficits occur in the three core areas of communication, social interaction, and behavior. The causes of autism are unknown, but clinical genetic studies show strong evidence in favor of a genetic etiology. Molecular genetic studies report some association with candidate genes, and candidate regions have emerged from several genome-wide linkage studies. Here we report a clinical case of autism with a deletion on chromosome 2 in a young male with high-functioning autism. The deletion seems to correspond with regions emerging from linkage studies. We propose this as a possible candidate region in the search for autism genes.

Loss of a single amino acid from dystrophin resulting in Duchenne muscular dystrophy with retention of dystrophin protein.

Almost all of the thousands of pathogenic mutations which have been described in the dystrophin gene either reduce protein production or remove large regions of the protein. This has severely limited the use of mutational information for the functional dissection of the dystrophin protein and increases the value of rare subtle mutations. We report a 3-bp deletion which removes a single highly conserved residue (glutamic acid 3367) adjacent to the dystrophin ZZ domain. This results in a phenotype of Duchenne muscular dystrophy with substantial retention of a presumably functionally compromised dystrophin protein. Two missense mutations (both affecting nearby residues) have been previously reported to result in this unusual combination of severe phenotype and high protein level. We discuss the functional implications of this and other mutations in the light of the predicted structure of the region. The pathogenicity of E3367del serves to emphasise the functional importance of this region of the dystrophin protein.

Dysmorphic features and learning disability in an adult male with pure partial trisomy 17q24-q25 due to a terminal duplication.

We describe an adult male with severe learning disability, epilepsy, and dysmorphic features. Cytogenetic studies demonstrated a terminal duplication of the long arm of chromosome 17, resulting in partial trisomy 17q24-q25. Our patient shows some of the characteristic features of the distal 17q phenotype, but in addition has more unusual features such as epilepsy, sensorineural hearing loss, and long fingers and overlapping toes. We suggest that these features occur with terminal duplications of 17q.