The effect of cell synchronization on the efficiency of stable gene transfer by electroporation

We synchronized thymidine kinase deficient mouse Ltk-cells by two different methods, hydroxyurea double-block treatment or aphidicolin single-block treatment and transformed them with the cloned herpes simplex virus thymidine kinase gene at various time intervals by the electroporation technique. Marked enhancement of stable transformation efficiency was observed at the time corresponding to the peak of G2/M phase. These results suggest that the G2/M phase is the most efficient period for stable gene transfer by electroporation.     

Structure-dependent variation in the mutagenic, prophage-inducing and antibacterial activities of 5-nitro-2-furamide derivatives

A comparative survey of the mutagenic, prophage-inducing and antibacterial activities of 3 structure-related series of 5-nitro-furan derivatives including 5-nitro-2-furohydrazide imide, 5-nitro-2-furamide oxime and 5-nitro-2-furohydrazide has been undertaken. Among the compounds assayed, the 5-nitro-2-furohydrazide imide series was found to be most active with regard to mutagenic and antibacterial activities against Salmonella typhimurium TA100 and prophage-inducing activity in Escherichia coli GY5027. A clear correlation was observed between the chemical structure and the mutagenic and prophage-inducing activities which were approximately correlated to the antibacterial activity.     

Bioactive compounds in plant materials for the prevention of diabetesand obesity

Plant materials have been widely studied for their preventive and therapeutic effects for type 2 diabetes mellitus (T2DM) and obesity. The effect of a plant material arises from its constituents, and the study of these bioactive compounds is important to achieve a deeper understanding of its effect at the molecular level. In particular, the study of the effects of such bioactive compounds on various biological processes, from digestion to cellular responses, is required to fully understand the overall effects of plant materials in these health contexts. In this review, I summarize the bioactive compounds we have recently studied in our research group that target digestive enzymes, dipeptidyl peptidase-4, myocyte glucose uptake, and lipid accumulation in adipocytes.

Abbreviations: AC: adenylyl cyclase; AMPK: AMP-activated protein kinase; βAR: β-adrenergic receptor; CA: catecholamine; cAMP: cyclic adenosine monophosphate; cGMP: cyclic guanosine monophosphate; DPP-4: dipeptidyl peptidase-4; ERK: extracellular signal-regulated kinase; GC: guanylyl cyclase; GH: growth hormone; GLP-1: glucagon-like peptide-1; GLUT: glucose transporter; HSL: hormone-sensitive lipase; IR: insulin receptor; IRS: insulin receptor substrate; MAPK: mitogen-activated protein kinase; MEK: MAPK/ERK kinase; MG: maltase-glucoamylase; NP: natriuretic peptide; NPR: natriuretic peptide receptor; mTORC2: mechanistic target of rapamycin complex-2; PC: proanthocyanidin; PI3K: phosphoinositide 3-kinase; PKA: cAMP-dependent protein kinase; PKB (AKT): protein kinase B; PKG: cGMP-dependent protein kinase; PPARγ: peroxisome proliferator-activated receptor-γ; SGLT1: sodium-dependent glucose transporter 1; SI: sucrase-isomaltase; T2DM: type 2 diabetes mellitus; TNFα: tumor necrosis factor-α.     

Analysis of the CK2-dependent phosphorylation of serine 13 in Cdc37 using a phospho-specific antibody and phospho-affinity gel electrophoresis

The CK2-dependent phosphorylation of Ser13 in cell division cycle protein 37 (Cdc37), a kinase-specific heat shock protein 90 (Hsp90) cochaperone, has previously been reported to be essential for the association of Cdc37 with signaling protein kinases [Bandhakavi S, McCann RO, Hanna DE & Glover CVC (2003) J Biol Chem278, 2829-2836; Shao J, Prince T, Hartson SD & Matts RL (2003) J Biol Chem278, 38117-38220; Miyata Y & Nishida E (2004) Mol Cell Biol24, 4065-4074]. Here we describe a new phospho-specific antibody against Cdc37 that recognizes recombinant purified Cdc37 only when incubated with CK2 in the presence of Mg(2+) and ATP. The replacement of Ser13 in Cdc37 by nonphosphorylatable amino acids abolished binding to this antibody. The antibody was specific for phosphorylated Cdc37 and did not crossreact with other CK2 substrates such as Hsp90 and FK506-binding protein 52. Using this antibody, we showed that complexes of Hsp90 with its client signaling kinases, Cdk4, MOK, v-Src, and Raf1, contained the CK2-phosphorylated form of Cdc37 in vivo. Immunofluorescent staining showed that Hsp90 and the phosphorylated form of Cdc37 accumulated in epidermal growth factor-induced membrane ruffles. We further characterized the phosphorylation of Cdc37 using phospho-affinity gel electrophoresis. Our analyses demonstrated that the CK2-dependent phosphorylation of Cdc37 on Ser13 caused a specific gel mobility shift, and that Cdc37 in the complexes between Hsp90 and its client signaling protein kinases was in the phosphorylated form. Our results show the physiological importance of CK2-dependent Cdc37 phosphorylation and the usefulness of phospho-affinity gel electrophoresis in protein phosphorylation analysis.     

Discrimination of reproductive forms of Thrips tabaci (Thysanoptera: Thripidae) by PCR with sequence specific primers

In agriculture, although it is important to identify species of pest insects, the morphological identification is often difficult. DNA genotyping is useful for the identification of species in morphologically indiscriminable species. Thrips tabaci (Lindeman) can be divided into two reproductive forms (arrhenotoky and thelytoky, each of which different in pesticide resistance) but morphological discrimination is not possible. Here, we establish a simple method to discriminate the strains based on their mitochondrial DNA sequences. Phylogenetic analysis including the T. tabaci and congeneric species provided ancestor sequences of each strain of T tabaci. Based on the ancestor sequences, we developed a primer set that include strain specific primers on sense strand and common primer on anti sense strand. Using this primer set, the strains of 196 individuals of T. tabaci were successfully assigned to each ofgenotypic forms. As the phylogeny and ancestor sequences were based on worldwide samples, this method will work well on most populations around the world.     

Experimental evaluation of birds as disseminators of the cosmopolitan tick Rhipicephalus sanguineus (Acari: Ixodidae)

Rhipicephalus sanguineus is believed to be the most widespread tick species of the world and its dissemination seems to rely on the diffusion of its main host, the dog. Empirical observations indicate that several bird species in urban areas regularly steal dog food. Such circumstances create a chance for R. sanguineus ticks to climb on birds and carry ticks to another site. In this work we evaluated experimentally the likelihood of birds (chicks) to either feed and/or carry R. sanguineus ticks from an infested site to another and to infest a host (rabbit) in the new location. Chicks were not suitable hosts for R. sanguineus ticks. Not a single adult tick engorged on chicks, yield as well as weight of engorged larvae and nymphs were very low and feeding period of these ticks was very long. However, a few larvae and, chiefly, nymphs were delivered to a new location either mechanically or after attachment and engorging total or partially on chicks. A few of these ticks fed successfully on rabbits. Further evidence on the capacity of birds to introduce R. sanguineus into non-infested dog settings should be provided by systematic examination of birds from urban areas, close to tick infested households.