THF peroxide as a factor in generating desulphurised products from the solid-phase synthesis of phosphorothioate-modified oligonucleotides.

Antisense oligonucleotides (ASOs) are generally obtained chemical synthesis on a solid support and phosphorothioate (PS) modification with a phosphate backbone to increase their stability and activity. However, desulphurised products, in which PS is partially replaced by phosphodiesters, are generally formed during the chemical synthesis of ASO and are difficult to separate from the desired PS-modified ASO by chromatography. Therefore, revealing the unknown factors that cause the formation of desulphurised products and proposing methods to inhibit their formation are highly desirable.

HiBiT-qIP, HiBiT-based quantitative immunoprecipitation, facilitates the determination of antibody affinity under immunoprecipitation conditions.

The affinity of an antibody for its antigen serves as a critical parameter for antibody evaluation. The evaluation of antibody-antigen affinity is essential for a successful antibody-based assay, particularly immunoprecipitation (IP), due to its strict dependency on antibody performance. However, the determination of antibody affinity or its quantitative determinant, the dissociation constant (K), under IP conditions is difficult.

Structure and function of the bi-directional bacterial flagellar motor.

The bacterial flagellum is a locomotive organelle that propels the bacterial cell body in liquid environments. The flagellum is a supramolecular complex composed of about 30 different proteins and consists of at least three parts: a rotary motor, a universal joint, and a helical filament. The flagellar motor of Escherichia coli and Salmonella enterica is powered by an inward-directed electrochemical potential difference of protons across the cytoplasmic membrane.

Polyamine sensitivity of gap junctions is required for skin pattern formation in zebrafish.

Gap junctions allow the direct and bidirectional transfer of small molecules between cells. Polyamine sensitivity, which has been observed for a certain gap junction in vitro, confers rectification property to gap junction. Here we report that the polyamine sensitivity of gap junctions in vivo is crucial for skin pattern formation in zebrafish.

Expression levels of Protocadherin-alpha transcripts are decreased by nonsense-mediated mRNA decay with frameshift mutations and by high DNA methylation in their promoter regions.

The mouse protocadherin (Pcdh) clusters, Pcdh-alpha, -beta, and -gamma, are located on chromosome 18. Many polymorphic variations are found in the Pcdh-alpha genes in wild-derived and laboratory mouse strains. In comparing the expression levels of Pcdh-alpha isoforms among several strains, we observed lower expression levels of Pcdh-alpha9 in BLG2 and BFM/2, and of Pcdh-alpha8 in C57BL/6 (B6) than in the other strains.

Split single-cell RT-PCR analysis of Purkinje cells.

This protocol details a method for analyzing the expression of multiple genes from a single Purkinje neuron, including the determination of whether the gene expression is monoallelic or biallelic. The protocol describes how to extract a single, living Purkinje cell for reverse transcription, divide the cDNAs into three equal samples and subject those to triplicate amplification of multiple targets by two rounds of PCR (first a multiplex PCR then a gene-specific nested PCR) and finally discriminate the allelic expression of the transcript by direct sequencing of the PCR products. In optimal conditions, this method permits the analysis of the expression of 18 genes in a single Purkinje cell.

Allelic gene regulation of Pcdh-alpha and Pcdh-gamma clusters involving both monoallelic and biallelic expression in single Purkinje cells.

The molecular basis for providing the identity and diversity of single neurons is a key for realizing the brain system. Diverse protocadherin isoforms encoded by the Pcdh-alpha and Pcdh-gamma gene clusters are expressed in all of the vertebrates studied. For the Pcdh-alpha isoforms, differential expression patterns have been found in single Purkinje cells by unusual monoallelic and combinatorial types of gene regulation.

Monoallelic yet combinatorial expression of variable exons of the protocadherin-alpha gene cluster in single neurons.

Diverse protocadherin-alpha genes (Pcdha, also called cadherin-related neuronal receptor or CNR) are expressed in the vertebrate brain. Their genomic organization involves multiple variable exons and a set of constant exons, similar to the immunoglobulin (Ig) and T-cell receptor (TCR) genes. This diversity can be used to distinguish neurons.

Genomic organization and transcripts of the zebrafish Protocadherin genes.

We have examined the protocadherin (Pcdh) gene clusters of the zebrafish (Danio rerio). At least three sets of the Pcdh gene cluster were found in the zebrafish genome. Here, we describe the complete organization of the DrPcdh2 gene clusters.

Transcription factors for lens development assessed in vivo.

Lens-cell differentiation occurs at a fairly early stage of embryogenesis and results in very simple tissue architecture. These features allow the embryonic lens to provide a paradigm of tissue development starting from tissue induction to tissue maturation. Not only have a number of transcription factors participating in lens development been identified but their actual functions are now assessed by modern approaches utilizing genetic and tissue manipulations of embryos.