Decreased ZO1 expression causes loss of time-dependent tight junction function in the liver of ob/ob mice.

Diabetes patients are at a high risk of developing complications related to angiopathy and disruption of the signal transduction system. The liver is one of the multiple organs damaged during diabetes. Few studies have evaluated the morphological effects of adhesion factors in diabetic liver. The influence of diurnal variation has been observed in the expression and functioning of adhesion molecules to maintain tissue homeostasis associated with nutrient uptake. The present study demonstrated that the rhythm-influenced functioning of tight junction was impaired in the liver of ob/ob mice. The tight junctions of hepatocytes were loosened during the dark period in control mice compared to those in ob/ob mice, where the hepatocyte gaps remained open throughout the day. The time-dependent expression of zonula occludens 1 (ZO1, encoded by Tjp1 gene) in the liver plays a vital role in the functioning of the tight junction. The time-dependent expression of ZO1 was nullified and its expression was attenuated in the liver of ob/ob mice. ZO1 expression was inhibited at the mRNA and protein levels. The expression rhythm of ZO1 was found to be regulated by heat shock factor (HSF)1/2, the expression of which was reduced in the liver of ob/ob mice. The DNA-binding ability of HSF1/2 was decreased in the liver of ob/ob mice compared to that in control mice. These findings suggest the involvement of impaired expression and functioning of adhesion factors in diabetic liver complications.

Structure-activity relationship studies on acremomannolipin A, the potent calcium signal modulator with a novel glycolipid structure 3: role of the length of alditol side chain.

Five homologs of a novel glycolipid acremomannolipin A (1a), the potential Ca(2+) signal modulator isolated from Acremonium strictum, bearing alditols of different length (1g-1k) were synthesized by a stereoselective ß-mannosylation of appropriately protected mannosyl sulfoxide (2) with five alditols (1g: C2, 1h: C3, 1i: C4, 1j: C5 and 1k: C7 units), and their potential in modulating Ca(2+) signaling were evaluated. Homologs with alditols of more than 4 carbons (1i, 1j and 1k) were equally or more potent than the parent compound (1a) regardless of the length of the alditol chain. Whereas activities of two homologs with shorter chains (1g and 1h) decreased to a considerable extent. The results indicated that the length of the alditol side chain was a crucial determinant for the potent calcium signal modulating activity.

Structure-activity relationship studies on acremomannolipin A, the potent calcium signal modulator with a novel glycolipid structure 2: Role of the alditol side chain stereochemistry.

Five alditol analogs 1b-1f of a novel glycolipid acremomannolipin A (1a), the potential Ca(2+) signal modulator isolated from Acremonium strictum, were synthesized by employing a stereoselective ß-mannosylation of appropriately protected mannose with five hexitols with different stereochemistry, and their potential on modulating Ca(2+) signaling were evaluated. All these analogs were more potent compared to the original compound 1a, and proved that mannitol stereochemistry of 1a was not critical for the potent calcium signal modulating.

Estimation of single-nucleotide polymorphism allele frequency by alternately binding probe competitive polymerase chain reaction.

Estimation of single-nucleotide polymorphism (SNP) allele frequency in pooled DNA samples is a promising approach to clarify the relationships between SNPs and diseases. Here, we present a simple, accurate, and cost-effective method for estimating SNP allele frequency, called alternately binding probe (ABProbe) competitive polymerase chain reaction (ABC-PCR) that entails no expensive devices for real-time fluorescence measurement and complex post-PCR steps. We prepared DNA pools of PCR products derived from homozygous samples of three different SNPs (ALDH2, GNB3, and HTR2A) in different portions, and the allele frequencies of these samples were estimated by ABC-PCR. Two alleles were coamplified by PCR with a fluorescent probe that binds to either alleles, and then fluorescence intensity was measured using a simple fluorometer. The ratio of the two alleles was calculated from the fluorescence intensity of the probe at the end-point. The estimated allele frequencies strongly correlated to the expected ratios for all three SNPs with high accuracy. When the allele frequencies were more than 5%, the relative standard deviations (R.S.D.s) of ABC-PCR were less than 20%. Moreover, we also confirmed that this method was applicable to SNP genotyping.

Calibration-curve-free quantitative PCR: a quantitative method for specific nucleic acid sequences without using calibration curves.

We have developed a simple quantitative method for specific nucleic acid sequences without using calibration curves. This method is based on the combined use of competitive polymerase chain reaction (PCR) and fluorescence quenching. We amplified a gene of interest (target) from DNA samples and an internal standard (competitor) with a sequence-specific fluorescent probe using PCR and measured the fluorescence intensities before and after PCR. The fluorescence of the probe is quenched on hybridization with the target by guanine bases, whereas the fluorescence is not quenched on hybridization with the competitor. Therefore, quench rate (i.e., fluorescence intensity after PCR divided by fluorescence intensity before PCR) is always proportional to the ratio of the target to the competitor. Consequently, we can calculate the ratio from quench rate without using a calibration curve and then calculate the initial copy number of the target from the ratio and the initial copy number of the competitor. We successfully quantified the copy number of a recombinant DNA of genetically modified (GM) soybean and estimated the GM soybean contents. This method will be particularly useful for rapid field tests of the specific gene contamination in samples.