Development of AC microelectrophoresis for rapid protein affinity evaluation

We have developed a new method for evaluating the affinity interactions between two different proteins by applying an alternating current (AC) voltage to a micro-flow channel. An AC voltage was applied to the protein-modified microspheres in the micro-flow channel, which resulted in the oscillation of the microspheres owing to their surface charges. The oscillation amplitude showed a linear relationship with the charge density of the microspheres. As an example for protein affinity measurement, the amplitude changes of a profilin-modified microsphere were measured by the addition of actin. In the same electrical condition, the oscillation amplitude of the profilin-modified microsphere increased by ≈175% by binding with actin. Similar results in the principle were obtained for the affinity interaction between biotin and streptavidin. The results showed that the higher the charge density of the microspheres induced by binding with different proteins, the higher the oscillation amplitude of the microspheres, thus, suggesting a possible application of the micro-flow channel and AC voltage on the protein property study, as well as on the biosensor application using the oscillation amplitude changes.     

Improvement of the transactivation activity of phenylpropanoic acid-type peroxisome proliferator-activated receptor pan agonists: effect of introduction of fluorine at the linker part

We developed a potent peroxisome proliferator-activated receptor pan agonist (a candidate drug for treatment of altered metabolic homeostasis) by introducing fluorine atoms at appropriate position(s) of the known phenylpropionic acid-type pan agonist TIPP-703.     

4-(Anilino)pyrrole-2-carboxamides: Novel non-steroidal/non-anilide type androgen antagonists effective upon human prostate tumor LNCaP cells with mutated nuclear androgen receptor

Various 4-(anilino)pyrrole-2-carboxamides were designed and synthesized as novel androgen receptor (AR) antagonists without steroidal or anilide structure, based on our strategy for developing full antagonists of nuclear receptors. Introduction of a bulky N-alkyl group, such as a cyclohexylmethyl or benzyl group, increased the binding affinity for wild-type AR and the potency for growth inhibition of androgen-dependent SC-3 cells. Among the compounds obtained, N-[4-[(benzyl)(4-nitrophenyl)amino]-1-methylpyrrole-2-carbonyl]pyrrolidine (22) is as potent an AR antagonist as the typical anilide-type AR antagonists hydroxyflutamide and bicalutamide. Further, compound 22 had potent binding affinity for T877A mutated AR, and dose-dependently inhibited the testosterone-induced production of prostate-specific antigen in LNCaP cells bearing T877A AR.     

Synthesis and absolute structures of Mycoplasma pneumoniae β-glyceroglycolipid antigens

Just recently, a pair of β-glycolipids was isolated from the cell membrane of Mycoplasma pneumoniae as a mixture of the two compounds. They are the major immunodeterminants of this pathogenic Mycoplasma and indicate high medicinal potential. They have a β-(1→6)-linked disaccharide structure close to each other; one has β-d-galactopyranoside (β-Gal-type 1) at the non-reducing terminal, and another has β-d-glucopyranoside (β-Glc-type 2). In the present study, the first stereoselective synthesis was conducted for each of the two β-glycolipids 1 and 2. ¹H NMR and TLC-immunostaining studies of the synthetic compounds enable us to establish the absolute structures having the β-(1→6)-linked disaccharides at the glycerol sn-3 position.     

Anatomical and histological profiling of orphan G-protein-coupled receptor expression in gastrointestinal tract of C57BL/6J mice

G-protein-coupled receptors (GPCRs) constitute the largest family of transmembrane receptors and regulate a variety of physiological and disease processes. Although the roles of many non-odorant GPCRs have been identified in vivo, several GPCRs remain orphans (oGPCRs). The gastrointestinal (GI) tract is the largest endocrine organ and is a promising target for drug discovery. Given their close link to physiological function, the anatomical and histological expression profiles of benchmark GI-related GPCRs, such as the cholecystokinin-1 receptor and GPR120, and 106 oGPCRs were investigated in the mucosal and muscle-myenteric nerve layers in the GI tract of C57BL/6J mice by quantitative real-time polymerase chain reaction. The mRNA expression patterns of these benchmark molecules were consistent with previous in situ hybridization and immunohistochemical studies, validating the experimental protocols in this study. Of 96 oGPCRs with significant mRNA expression in the GI tract, several oGPCRs showed unique expression patterns. GPR85, GPR37, GPR37L1, brain-specific angiogenesis inhibitor (BAI) 1, BAI2, BAI3, and GPRC5B mRNAs were preferentially expressed in the muscle-myenteric nerve layer, similar to GPCRs that are expressed in both the central and enteric nerve systems and that play multiple regulatory roles throughout the gut-brain axis. In contrast, GPR112, trace amine-associated receptor (TAAR) 1, TAAR2, and GPRC5A mRNAs were preferentially expressed in the mucosal layer, suggesting their potential roles in the regulation of secretion, immunity, and epithelial homeostasis. These anatomical and histological mRNA expression profiles of oGPCRs provide useful clues about the physiological roles of oGPCRs in the GI tract.     

Polymorphism distribution and structural conservation in RNA-sensing Toll-like receptors 3, 7, and 8 in pigs

Background

Viral genomic RNA—both single-stranded (ss) and double-stranded (ds)—is recognized by RNA-sensing Toll-like receptors (TLRs), notably TLR3 (dsRNA), TLR7 (ssRNA), and TLR8 (ssRNA). However, our knowledge of the roles of porcine TLR3, 7, and 8 in antiviral immunity is inadequate.

Methods

From information on exon–intron boundaries obtained through comparisons of the genomic and cDNA sequences, polymorphisms in the coding sequences of each gene were detected in 84 male pigs of 11 breeds.

Results

Genomic structures are conserved between pigs and humans. The RNA-sensing TLR genes had fewer polymorphisms causing amino acid alterations than did the cell-surface TLR genes, but the alterations were distributed with a similar bias toward ectodomains.

Conclusions

The low level of diversity of substitutive polymorphisms in RNA-sensing TLRs than cell-surface ones implies that polymorphisms severely affecting function have been eliminated by selection pressure during longstanding pig breeding.

General significance

Recognition of virus-derived RNA is critical in host defense against infection. These results should provide a useful clue to analysis of the association between polymorphisms in RNA-sensing TLRs and disease resistance.     

Difference in number of loci of swine leukocyte antigen classical class I genes among haplotypes

The structure of the entire genomic region of swine leukocyte antigen (SLA)-the porcine major histocompatibility complex--was recently elucidated in a particular haplotype named Hp-1.0 (H01). However, it has been suggested that there are differences in the number of loci of SLA genes, particularly classical class I genes, among haplotypes. To clarify the between-haplotype copy number variance in genes of the SLA region, we sequenced the genomic region carrying SLA classical class I genes on two different haplotypes, revealing increments of up to six in the number of classical class I genes in a single haplotype. All of the SLA-1(-like) (SLA-1 and newly designated SLA-12) and SLA-3 genes detected in the haplotypes thus analyzed were transcribed in the individual. The process by which duplication of SLA classical class I genes was likely to have occurred was interpreted from an analysis of repetitive sequences adjacent to the duplicated class I genes.     

Voltage-gated proton channel is expressed on phagosomes

Voltage-gated proton channel has been suggested to help NADPH oxidase activity during respiratory burst of phagocytes through its activities of compensating charge imbalance and regulation of pH. In phagocytes, robust production of reactive oxygen species occurs in closed membrane compartments, which are called phagosomes. However, direct evidence for the presence of voltage-gated proton channels in phagosome has been lacking. In this study, the expression of voltage-gated proton channels was studied by Western blot with the antibody specific to the voltage-sensor domain protein, VSOP/Hv1, that has recently been identified as the molecular correlate for the voltage-gated proton channel. Phagosomal membranes of neutrophils contain VSOP/Hv1 in accordance with subunits of NADPH oxidases, gp91, p22, p47 and p67. Superoxide anion production upon PMA activation was significantly reduced in neutrophils from VSOP/Hv1 knockout mice. These are consistent with the idea that voltage-gated proton channels help NADPH oxidase in phagocytes to produce reactive oxygen species.