The role of the helper lipid dioleoylphosphatidylethanolamine (DOPE) for DNA transfection cooperating with a cationic lipid bearing ethylenediamine

Gene therapy is expected to treat various incurable diseases including viral infections, autoimmune disorders, and cancers. Cationic lipids (CL) have been used as carriers of therapeutic DNAs for gene therapy because they can form a complex with DNA and such a complex can be incorporated into cells and transport the bound DNA to cytosol. The CL/DNA complexes are called lipoplexes and categorized as a non-viral vector. Lipoplexes are often prepared by adding a neutral phospholipid dioleoylphosphatidylethanolamine (DOPE) to CL in order to enhance transfection. However, the role of DOPE is not fully understood. We synthesized a new CL having an ethylenediamine cationic head group, denoted by DA, and found that addition of DOPE to DA achieved a good efficiency, almost in the similar level of commonly used transfection reagent Lipofectamine 2000 (Invitrogen). The composition of DA:DOPE = 1:1 showed the highest efficiency. This lipoplex showed structural transition when pH was changed from 7 to 4, corresponding pH lowering in late endosome, while DOPE itself showed structural transition at more basic pH around 8. The present data showed that the DOPE/DA composition determines the structural transition pH and choosing a suitable pH, i.e., a suitable composition, is essential to increase the transfection efficiency.     

Inhibition of HIV-1 replication by a tricyclic coumarin GUT-70 in acutely and chronically infected cells

The anti-HIV-1 activity of GUT-70, a natural product derived from the stem bark of Chlophyllum brasiliense, was evaluated. GUT-70 inhibited HIV-1 replication in both acutely and chronically infected cells through suppression of NF-κB. Our results strengthen the idea that NF-κB pathway is one of the potential targets to control HIV-1 replication and that GUT-70 could serve as a lead compound to develop novel therapeutic agents against HIV-1 infection.     

Synthesis and evaluation of N-alkyl-S-[3-(piperidin-1-yl)propyl]isothioureas: High affinity and human/rat species-selective histamine H3 receptor antagonists

S-Alkyl-N-alkylisothiourea compounds containing various cyclic amines were synthesized in the search for novel nonimidazole histamine H₃ receptor (H₃R) antagonists. Among them, four N-alkyl S-[3-(piperidin-1-yl)propyl]isothioureas 18, 19, 22, and 23 were found to exhibit potent and selective H₃R antagonistic activities against in vitro human H₃R, but were inactive against in vitro human H₄R. Furthermore, three alkyl homologs 18–20 showed inactivity for histamine release in in vivo rat brain microdialysis, suggesting differences in antagonist affinities between species. In addition, in silico docking studies of N-[4-(4-chlorophenyl)butyl]-S-[3-piperidin-1-yl)propyl]isothiourea 19 and a shorter homolog 17 with human/rat H₃Rs revealed that structural differences between the antagonist-docking cavities of rat and human H₃Rs were likely caused by the Ala122/Val122 mutation.     

Characterization of the receptor binding residues of kisspeptins by positional scanning using peptide photoaffinity probes

Kisspeptins, endogenous peptide ligands for GPR54, play an important role in GnRH secretion. Since in vivo administration of kisspeptins induces increased plasma LH levels, GPR54 agonists hold promise as therapeutic agents for the treatment of hormonal secretion diseases. To facilitate the design of novel potent GPR54 ligands, residues in kisspeptins that involve in the interaction with GPR54 were investigated by kisspeptin-based photoaffinity probes. Herein, we report the design and synthesis of novel kisspeptin-based photoaffinity probes, and the application to crosslinking experiments for GPR54-expressing cells.     

Design and synthesis of biotin- or alkyne-conjugated photoaffinity probes for studying the target molecules of PD 404182

To investigate the mechanism of action of the potent antiviral compound PD 404182, three novel photoaffinity probes equipped with a biotin or alkyne indicator were designed and synthesized based on previous structure–activity relationship studies. These probes retained the potent anti-HIV activity of the original pyrimidobenzothiazine derivatives. In photoaffinity labeling studies using HIV-1-infected H9 cells (H9IIIB), eight potential proteins were observed to bind PD 404182.     

Mutation strategies for obtaining chitooligosaccharides with longer chains by transglycosylation reaction of family GH18 chitinase

Enhancing the transglycosylation (TG) activity of glycoside hydrolases does not always result in the production of oligosaccharides with longer chains, because the TG products are often decomposed into shorter oligosaccharides. Here, we investigated the mutation strategies for obtaining chitooligosaccharides with longer chains by means of TG reaction catalyzed by family GH18 chitinase A from Vibrio harveyi (VhChiA). HPLC analysis of the TG products from incubation of chitooligosaccharide substrates, GlcNAc_n, with several mutant VhChiAs suggested that mutant W570G (mutation of Trp570 to Gly) and mutant D392N (mutation of Asp392 to Asn) significantly enhanced TG activity, but the TG products were immediately hydrolyzed into shorter GlcNAc_n. On the other hand, the TG products obtained from mutants D313A and D313N (mutations of Asp313 to Ala and Asn, respectively) were not further hydrolyzed, leading to the accumulation of oligosaccharides with longer chains. The data obtained from the mutant VhChiAs suggested that mutations of Asp313, the middle aspartic acid residue of the DxDxE catalytic motif, to Ala and Asn are most effective for obtaining chitooligosaccharides with longer chains.     

No Association of Coffee Consumption with Gastric Ulcer,Duodenal Ulcer,Reflux Esophagitis,and Non-Erosive Reflux Disease: A Cross-Sectional Study of 8,013 Healthy Subjects in Japan

Probably due to caffeine-induced gastric acid secretion, negative effects of coffee upon various upper-gastrointestinal diseases have been precariously accepted, despite the inadequate epidemiological evidence. Our aim is to evaluate the effect of coffee consumption on four major acid-related diseases: gastric ulcer (GU), duodenal ulcer (DU), reflux esophagitis (RE), and non-erosive reflux disease (NERD) based on the large-scale multivariate analysis. Of the 9,517 healthy adults, GU, DU, and RE were diagnosed by endoscopy, and NERD was diagnosed by the symptoms of heartburn and regurgitation without esophageal erosion. Associations between coffee consumption and the four disorders were evaluated, together with age, gender, body mass index (BMI), Helicobacter pylori (HP) infection status, pepsinogen I/II ratio, smoking, and alcohol. We further performed meta-analysis using the random effects model to redefine the relationship between coffee intake and peptic ulcer disease. The eligible 8,013 study subjects comprised of 5,451 coffee drinkers and 2,562 non-coffee drinkers. By univariate analysis, age, BMI, pepsinogen I/II ratio, smoking, and alcohol showed significant associations with coffee consumption. By multiple logistic regression analysis, positively correlated factors with significance were HP infection, current smoking, BMI, and pepsinogen I/II ratio for GU; HP infection, pepsinogen I/II ratio, and current smoking for DU; HP non-infection, male, BMI, pepsinogen I/II ratio, smoking, age, and alcohol for RE; younger age, smoking, and female for NERD. The meta-analyses could detect any association of coffee consumption with neither GU nor DU. In conclusion, there are no significant relationship between coffee consumption and the four major acid-related upper gastrointestinal disorders.     

Background Factors of Reflux Esophagitis and Non-Erosive Reflux Disease: A Cross-Sectional Study of 10,837 Subjects in Japan

Background

Despite the high prevalence of gastroesophageal reflux disease (GERD), its risk factors are still a subject of controversy. This is probably due to inadequate distinction between reflux esophagitis (RE) and non-erosive reflux disease (NERD), and is also due to inadequate evaluation of adjacent stomach. Our aim is therefore to define background factors of RE and NERD independently, based on the evaluation of Helicobacter pylori infection and gastric atrophy.

Methods

We analyzed 10,837 healthy Japanese subjects (6,332 men and 4,505 women, aged 20–87 years) who underwent upper gastrointestinal endoscopy. RE was diagnosed as the presence of mucosal break, and NERD was diagnosed as the presence of heartburn and/or acid regurgitation in RE-free subjects. Using GERD-free subjects as control, background factors for RE and NERD were separately analyzed using logistic regression to evaluate standardized coefficients (SC), odds ratio (OR), and p-value.

Results

Of the 10,837 study subjects, we diagnosed 733 (6.8%) as RE and 1,722 (15.9%) as NERD. For RE, male gender (SC = 0.557, OR = 1.75), HP non-infection (SC = 0.552, OR = 1.74), higher pepsinogen I/II ratio (SC = 0.496, OR = 1.64), higher BMI (SC = 0.464, OR = 1.60), alcohol drinking (SC = 0.161, OR = 1.17), older age (SC = 0.148, OR = 1.16), and smoking (SC = 0.129, OR = 1.14) are positively correlated factors. For NERD, HP infection (SC = 0.106, OR = 1.11), female gender (SC = 0.099, OR = 1.10), younger age (SC = 0.099, OR = 1.10), higher pepsinogen I/II ratio (SC = 0.099, OR = 1.10), smoking (SC = 0.080, OR = 1.08), higher BMI (SC = 0.078, OR = 1.08), and alcohol drinking (SC = 0.076, OR = 1.08) are positively correlated factors. Prevalence of RE in subjects with chronic HP infection and successful HP eradication denotes significant difference (2.3% and 8.8%; p<0.0001), whereas that of NERD shows no difference (18.2% and 20.8%; p = 0.064).

Conclusions

Significantly associated factors of NERD are considerably different from those of RE, indicating that these two disorders are pathophysiologically distinct. Eradication of Helicobacter pylori may have disadvantageous effects on RE but not on NERD.     

miR-146a Polymorphism (rs2910164) Predicts Colorectal Cancer Patients’ Susceptibility to Liver Metastasis

miR-146a plays important roles in cancer as it directly targets NUMB, an inhibitor of Notch signaling. miR-146a is reportedly regulated by a G>C polymorphism (SNP; rs2910164). This polymorphism affects various cancers, including colorectal cancer (CRC). However, the clinical significance of miR-146a polymorphism in CRC remains unclear. A total of 59 patients with CRC were divided into 2 groups: a CC/CG genotype (n = 32) and a GG genotype (n = 27), based on the miR-146a polymorphism. cDNA microarray analysis was performed using 59 clinical samples. Significantly enriched gene sets in each genotype were extracted using GSEA. We also investigated the association between miR-146a polymorphism and miR-146a, NUMB expression or migratory response in CRC cell lines. The CC/CG genotype was associated with significantly more synchronous liver metastasis (p = 0.007). A heat map of the two genotypes showed that the expression profiles were clearly stratified. GSEA indicated that Notch signaling and JAK/STAT3 signaling were significantly associated with the CC/CG genotype (p = 0.004 and p = 0.023, respectively). CRC cell lines with the pre-miR-146a/C revealed significantly higher miR-146a expression (p = 0.034) and higher NUMB expression and chemotactic activity. In CRC, miR-146a polymorphism is involved in liver metastasis. Identification of this polymorphism could be useful to identify patients with a high risk of liver metastasis in CRC.