Specific Mutations Reverse Regulatory Effects of Adenosine Phosphates and Increase Their Binding Stoichiometry in CBS Domain-Containing Pyrophosphatase.

Regulatory cystathionine ß-synthase (CBS) domains are widespread in proteins; however, difficulty in structure determination prevents a comprehensive understanding of the underlying regulation mechanism. Tetrameric microbial inorganic pyrophosphatase containing such domains (CBS-PPase) is allosterically inhibited by AMP and ADP and activated by ATP and cell alarmones diadenosine polyphosphates. Each CBS-PPase subunit contains a pair of CBS domains but binds cooperatively to only one molecule of the mono-adenosine derivatives. We used site-directed mutagenesis of Desulfitobacterium hafniense CBS-PPase to identify the key elements determining the direction of the effect (activation or inhibition) and the "half-of-the-sites" ligand binding stoichiometry. Seven amino acid residues were selected in the CBS1 domain, based on the available X-ray structure of the regulatory domains, and substituted by alanine and other residues. The interaction of 11 CBS-PPase variants with the regulating ligands was characterized by activity measurements and isothermal titration calorimetry. Lys100 replacement reversed the effect of ADP from inhibition to activation, whereas Lys95 and Gly118 replacements made ADP an activator at low concentrations but an inhibitor at high concentrations. Replacement of these residues for alanine increased the stoichiometry of mono-adenosine phosphate binding by twofold. These findings identified several key protein residues and suggested a "two non-interacting pairs of interacting regulatory sites" concept in CBS-PPase regulation.

Assembly of Thiosubstituted Benzoxazoles via Copper-Catalyzed Coupling of Thiols with 5-Iodotriazoles Serving as Diazo Surrogates.

An efficient cascade approach to thiosubstituted benzoxazoles has been developed. The transformation starts with in situ generation of a diazo compound via annulation-triggered electrocyclic opening of the 1,2,3-triazole ring. The subsequent Cu-catalyzed trapping of diazo intermediates by various thiols affords the desired heterocycles in generally good yields of up to 91%. The protocol features very good functional group tolerance and is applicable to substrates with different electronic properties.

Evidence for high frequency of chromosomal mosaicism in spontaneous abortions revealed by interphase FISH analysis.

Numerical chromosomal imbalances are a common feature of spontaneous abortions. However, the incidence of mosaic forms of chromosomal abnormalities has not been evaluated. We have applied interphase multicolor fluorescence in situ hybridization using original DNA probes for chromosomes 1, 9, 13, 14, 15, 16, 18, 21, 22, X, and Y to study chromosomal abnormalities in 148 specimens of spontaneous abortions. We have detected chromosomal abnormalities in 89/148 (60.1%) of specimens. Among them, aneuploidy was detected in 74 samples (83.1%). In the remaining samples, polyploidy was detected. The mosaic forms of chromosome abnormality, including autosomal and sex chromosomal aneuploidies and polyploidy (31 and 12 cases, respectively), were observed in 43/89 (48.3%) of specimens. The most frequent mosaic form of aneuploidy was related to chromosome X (19 cases). The frequency of mosaic forms of chromosomal abnormalities in samples with male chromosomal complement was 50% (16/32 chromosomally abnormal), and in samples with female chromosomal complement, it was 47.4% (27/57 chromosomally abnormal). The present study demonstrates that the postzygotic or mitotic errors leading to chromosomal mosaicism in spontaneous abortions are more frequent than previously suspected. Chromosomal mosaicism may contribute significantly to both pregnancy complications and spontaneous fetal loss.