Tozo Nishiyama

Hypoglycemic effects of clove (Syzygium aromaticum flower buds) on genetically diabetic KK-Ay mice and identification of the active ingredients

Clove (Syzygium aromaticum flower buds) EtOH extract significantly suppressed an increase in blood glucose level in type 2 diabetic KK-Ay mice. In-vitro evaluation showed the extract had human peroxisome proliferator-activated receptor (PPAR)-γ ligand-binding activity in a GAL4-PPAR-γ chimera assay. Bioassay-guided fractionation of the EtOH extract resulted in the isolation of eight compounds, of which dehydrodieugenol (2) and dehydrodieugenol B (3) had potent PPAR-γ ligand-binding activities, whereas oleanolic acid (4), a major constituent in the EtOH extract, had moderate activity. Furthermore, 2 and 3 were shown to stimulate 3T3-L1 preadipocyte differentiation through PPAR-γ activation. These results indicate that clove has potential as a functional food ingredient for the prevention of type 2 diabetes and that 2–4 mainly contribute to its hypoglycemic effects via PPAR-γ activation.

Deletion of DNA ligase IV homolog confers higher gene targeting efficiency on homologous recombination in Komagataella phaffii

&NA; The non‐conventional yeast Komagataella phaffii, formerly Pichia pastoris, is a popular host for recombinant protein production. The relatively lower gene targeting efficiency observed in this species occurs due to high levels of non‐homologous recombination activity. In the current study, we explored the function of the K. phaffii homolog of DNA ligase IV (Dnl4p) by creating a DNL4‐disrupted strain. To assess the roles of non‐homologous end joining (NHEJ)‐related proteins in this species, strains deleted for either or both genes encoding Dnl4p or the telomeric Ku complex subunit (Ku70p) were generated. These deletions were constructed by either of two distinct marker‐recycling methods (yielding either a seamless gene deletion or a Cre‐loxP‐mediated gene deletion). The resulting dnl4‐ and/or ku70‐deleted K. phaffii strains were used to evaluate gene targeting efficiency in gene knock‐out and gene knock‐in experiments. The Dnl4p‐defective strain showed improved gene targeting efficiency for homologous recombination compared to the wild‐type and Ku70p‐deffective strains. The dnl4 ku70 double knock‐out strain exhibited a further improvement in gene targeting efficiency. Thus, the K. phaffii dnl4 and dnl4 ku70 deletion strains are expected to serve as useful platforms for functional analysis and strain development in this species.