Proteinase K is an activator for the male-dependent spermiogenesis pathway in Caenorhabditis elegans: Its application to pharmacological dissection of spermiogenesis.

Caenorhabditis elegans spermiogenesis involves spermatid activation into spermatozoa. Activation occurs through either SPE-8 class-dependent or class-independent pathways. Pronase (Pron) activates the SPE-8 class-dependent pathway, whereas no in vitro tools are available to stimulate the SPE-8 class-independent pathway. Thus, whether there is a functional relationship between these two pathways is currently unclear. In this study, we found that proteinase K (ProK) can activate the SPE-8 class-independent pathway. In vitro spermiogenesis assays using Pron and ProK suggested that SPE-8 class proteins act in the hermaphrodite- and male-dependent spermiogenesis pathways and that some spermatid proteins presumably working downstream of spermiogenesis pathways, including MAP kinases, are preferentially involved in the SPE-8 class-dependent pathway. We screened a library of chemicals, and a compound that we named DDI-1 inhibited both Pron- and ProK-induced spermiogenesis. To our surprise, several DDI-1 analogues that are structurally similar to DDI-1 blocked Pron, but not ProK, induced spermiogenesis. Although the mechanism by which DDI-1 blocks spermiogenesis is yet unknown, we have begun to address this issue by selecting two DDI-1-resistant mutants. Collectively, our data support a model in which C. elegans male and hermaphrodite spermiogenesis each has its own distinct, parallel pathway.

Synthesis of Small Fluorescent Molecules and Evaluation of Photophysical Properties.

A series of aniline-based fluorophores were newly synthesized. To increase their fluorescence quantum yields, it was particularly important to substitute 3,3,3-trifluoroprop-1-enyl (TFPE) groups next to the amino group to benefit from an extended π-electron delocalization. Among these, 5-CN-2-TFPE-aniline was found to behave as an excellent fluorophore with a reasonable fluorescence quantum yield of 0.89 even in aqueous solution. l-Alanine peptide, a nonfluorescent analogue of 5-CN-2-TFPE-aniline, was synthesized and successfully employed as an enzyme probe to detect aminopeptidase N activity.

Development of a fluorogenic small substrate for dipeptidyl peptidase-4.

A series of aniline and m-phenylenediamine derivatives with electron-withdrawing 3,3,3-trifluoropropenyl substituents were synthesized as small and chemically stable fluorescent organic compounds. Their fluorescence performances were evaluated by converting 2,4-disubstituted aniline 1 to the non-fluorescent dipeptide analogue H-Gly-Pro-1 for the use as a fluorogenic substrate for dipeptidyl peptidase-4 (DPP-4). The progress of the enzymatic hydrolysis of H-Gly-Pro-1 with DPP-4 was monitored by fluorometric determination of 1 released into the reaction medium. The results suggest that 1 could be used as fluorophore in OFF-ON-type fluorogenic probes.

Crystal Structure of (E)-2-(3,3,3-tri-fluoro-prop-1-en-1-yl)aniline.

The mol-ecule of the title compound, C9H8F3N, adopts an E configuration with respect to the C=C double bond. The dihedral angle between the benzene ring and the prop-1-enyl group is 25.4 (3)°. In the crystal, mol-ecules are linked via pairs of N-H⋯F hydrogen bonds into inversion dimers with an R 2 2(16) ring motif. The dimers are linked by C-H⋯N hydrogen bonds, forming a ribbon structure along the b-axis direction. The ribbons are linked by N-H⋯π and C-H⋯π inter-actions, generating a three-dimensional network.