Development of an ON/OFF switchable fluorescent probe targeting His tag fused proteins in living cells

The fluorescent labeling of target proteins is useful for analyzing their functions and localization in cells, and several fluorescent probes have been developed. However, the fusion of tags such as green fluorescent protein (GFP) to target proteins occasionally affects their functions and/or localization in living cells. Therefore, an imaging method that uses short peptide tags such as hexa-histidine (the His tag) has been attracting increasing attention. Few studies have investigated ON/OFF switchable fluorescent probes for intracellular His-tagged proteins. We herein developed a novel ON/OFF switchable probe for imaging targeted intracellular proteins fused with a CH6 tag, which is composed of one cysteine residue and six histidine residues.     

New fluorogenic substrates for horseradish peroxidase: Rapid and sensitive assays for hydrogen peroxide and the peroxidase

p-Hydroxyphenyl compounds [3-(p-hydroxyphenyl)propionic acid, p-hydroxyphenethyl alcohol, hordenine, p-ethylphenol, 3-(p-hydroxyphenyl)-1-propanol, p-n-propylphenol, and p-hydroxyphenyllactic acid] were recently found to be excellent fluorogenic substrates for the horseradish peroxidase-mediated reaction with hydrogen peroxide. A very rapid and sensitive method for the fluorometric assays of hydrogen peroxide and the peroxidase was established by using 3-(p-hydroxyphenyl)propionic acid as the best of these substrates; hydrogen peroxide can be assayed precisely in amounts as small as 0.1 nmol, with peroxidase activity as low as 7.8 μU.     

Glycosylation of the OMP85 homolog of Porphyromonas gingivalis and its involvement in biofilm formation

OMP85 is a highly conserved outer membrane protein in all Gram-negative bacteria. We studied an uncharacterized OMP85 homolog of Porphyromonas gingivalis, a primary periodontal pathogen forming subgingival plaque biofilms. Using an outer-loop peptide antibody specific for the OMP85 of P. gingivalis, loop-3 Ab, we found a difference in the mobility of OMP85 on SDS-PAGE gel between the P. gingivalis wild-type and the isogenic galE mutant, a deglycosylated strain, suggesting that OMP85 naturally exists in a glycosylated form. This was also supported by a shift in OMP85 PAGE mobility after chemical deglycosylation treatment. Further, loop-3 Ab cross-reacted with the galE mutant stronger than the wild-type strain; and could inhibit biofilm formation in the galE mutant more than in the wild-type strain. In conclusion, this is the first report providing the evidence of OMP85 glycosylation and the involvement of OMP85 in biofilm formation.     

Rat mutations cvd and hob with cerebellar malformations map to chromosome 2.

In this paper, we executed genome mapping and comparative mapping analyses for cvd and hob, autosomal recessive mutations with cerebellar vermis defect and cerebellar dysplasia in the rat. For the linkage analysis, we produced three sets of backcross progeny, (ACI x CVD)F(1) and (F344 x CVD)F(1) females crossed to a cvd homozygous male rat, and (HOB x WKY)F(1) males crossed to hob homozygous female rats. Analysis of the segregation patterns of simple sequence length polymorphism (SSLP) markers scanning the whole rat genome allowed the mapping of these autosomal recessive mutations to rat Chromosome (Chr) 2. The most likely gene order is D2Mgh12 - D2Rat86 - D2Mit15 - D2Rat185 - cvd - D2Rat66 - D2Mgh13, and D2Mit18 - Fga -D2Mit14 - D2Rat16 - hob - D2Mgh13. Crossing test between a proven cvd heterozygous and a hob heterozygous rats demonstrated their allelism. Furthermore, comparative mapping indicated the cvd locus corresponds to mouse chromosome 3 and a strong candidate gene Unc5h3, a causative gene for the rostral cerebellar malformation mouse, was implicated.     

Osteocyte network; a negative regulatory system for bone mass augmented by the induction of Rankl in osteoblasts and Sost in osteocytes at unloading

Reduced mechanical stress is a major cause of osteoporosis in the elderly, and the osteocyte network, which comprises a communication system through processes and canaliculi throughout bone, is thought to be a mechanosensor and mechanotransduction system; however, the functions of osteocytes are still controversial and remain to be clarified. Unexpectedly, we found that overexpression of BCL2 in osteoblasts eventually caused osteocyte apoptosis. Osteoblast and osteoclast differentiation were unaffected by BCL2 transgene in vitro. However, the cortical bone mass increased due to enhanced osteoblast function and suppressed osteoclastogenesis at 4 months of age, when the frequency of TUNEL-positive lacunae reached 75%. In the unloaded condition, the trabecular bone mass decreased in both wild-type and BCL2 transgenic mice at 6 weeks of age, while it decreased due to impaired osteoblast function and enhanced osteoclastogenesis in wild-type mice but not in BCL2 transgenic mice at 4 months of age. Rankl and Opg were highly expressed in osteocytes, but Rankl expression in osteoblasts but not in osteocytes was increased at unloading in wild-type mice but not in BCL2 transgenic mice at 4 months of age. Sost was locally induced at unloading in wild-type mice but not in BCL2 transgenic mice, and the dissemination of Sost was severely interrupted in BCL2 transgenic mice, showing the severely impaired osteocyte network. These findings indicate that the osteocyte network is required for the upregulation of Rankl in osteoblasts and Sost in osteocytes in the unloaded condition. These findings suggest that the osteocyte network negatively regulate bone mass by inhibiting osteoblast function and activating osteoclastogenesis, and these functions are augmented in the unloaded condition at least partly through the upregulation of Rankl expression in osteoblasts and that of Sost in osteocytes, although it cannot be excluded that low BCL2 transgene expression in osteoblasts contributed to the enhanced osteoblast function.     

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.     

Two new phenolic glucosides and an ellagitannin from the leaves of Castanopsis sclerophylla

In the course of a chemotaxonomical study of Castanopsis species (Fagaceae), detailed investigation of the leaves of C. sclerophylla led to isolation of three new phenolic compounds together with 62 known compounds. The structures of the new compounds were elucidated as 2-O-galloyl-O-4,6-(S)-valoneoyl-d-glucose (1), 6-O-galloyl-1-O-vanilloyl-β-d-glucose (2), and 4″-O-galloylchestanin (3) by means of spectroscopic analyses and enzymatic hydrolysis with tannase. Comparison with other Castanopsis species indicated that C. sclerophylla characteristically accumulates chlorogenic acid and a dimeric ellagitannin, rugosin E. Triterpene hexahydroxydiphenoyl esters, which are major constituents of C. cuspidata var. sieboldii, C. hystrix, and C. fissa were not detected.