Identification and Characterization of a Novel Galactofuranose-Specific β-D-Galactofuranosidase from Streptomyces Species

β-D-galactofuranose (Galf) is a component of polysaccharides and glycoconjugates and its transferase has been well analyzed. However, no β-D-galactofuranosidase (Galf-ase) gene has been identified in any organism. To search for a Galf-ase gene we screened soil samples and discovered a strain, identified as a Streptomyces species by the 16S ribosomal RNA gene analysis, that exhibits Galf-ase activity for 4-nitrophenyl β-D-galactofuranoside (pNP-β-D-Galf) in culture supernatants. By draft genome sequencing of the strain, named JHA19, we found four candidate genes encoding Galf-ases. Using recombinant proteins expressed in Escherichia coli, we found that three out of four candidates displayed the activity of not only Galf-ase but also α-L-arabinofuranosidase (Araf-ase), whereas the other one showed only the Galf-ase activity. This novel Galf-specific hydrolase is encoded by ORF1110 and has an optimum pH of 5.5 and a Km of 4.4 mM for the substrate pNP-β-D-Galf. In addition, this enzyme was able to release galactose residue from galactomannan prepared from the filamentous fungus Aspergillus fumigatus, suggesting that natural polysaccharides could be also substrates. By the BLAST search using the amino acid sequence of ORF1110 Galf-ase, we found that there are homolog genes in both prokaryotes and eukaryotes, indicating that Galf-specific Galf-ases widely exist in microorganisms.     

Proxisome proliferator-activated receptor-α mediates high-fat, diet-enhanced daily oscillation of plasminogen activator inhibitor-1 activity in mice

Acute thrombotic events frequently occur in the early morning among hyperlipidemic patients. The activity of plasminogen activator inhibitor-1 (PAI-1), a potent inhibitor of the fibrinolytic system, oscillates daily, and this is considered one mechanism that underlies the morning onset of acute thrombotic events in hyperlipidemia. Although several studies have reported the expression of the PAI-1 gene is under the control of the circadian clock system, the molecular mechanism of the circadian transactivation of PAI-1 gene under hyperlipidemic conditions remains to be elucidated. Here, the authors investigated whether hyperlipidemia induced by a high-fat diet (HFD) enhances the daily oscillation of plasma PAI-1 activity in mice. The mRNA levels of the PAI-1 gene were increased and rhythmically fluctuated with high-oscillation amplitude in the livers of wild-type mice fed with the HFD. Circadian expression of proxisome proliferator-activated receptor-α (PPARα) mRNA was also augmented as well as that of PAI-1. Chromatin immunoprecipitation showed the HFD-induced hyperlipidemia significantly increased the binding of PPARα to the PAI-1 promoter. Luciferase reporter analysis using primary hepatocytes revealed CLOCK/BMAL1-mediated PAI-1 promoter activity was synergistically enhanced by cotransfection with PPARα/retinoid X receptor-α (RXRα), and this synergistic transactivation was repressed by negative limbs of the circadian clock, PERIOD2 and CRYPTOCHROME1. As expected, HFD-induced PAI-1 mRNA expression was significantly attenuated in PPARα-null mice. These results suggest a molecular link between the circadian clock and lipid metabolism system in the regulation of PAI-1 gene expression, and provide an aid for understanding why hyperlipidemia increases the risk of acute thrombotic events in the morning.     

Differences in accumulation of anthracyclines daunorubicin, doxorubicin and epirubicin in rat tissues revealed by immunocytochemistry

Although anthracycline antibiotics daunorubicin (DR), doxorubicin (DX), and epirubicin (ER) possess minor differences in their chemical structures, large differences are noted in their clinical use, as well as in cellular and plasma pharmacokinetic parameters in vivo. Immunocytochemistry for DR, DX, or ER was developed using an anti-DR monoclonal antibody (ADM-1-11), which has been demonstrated to react equally well with each of the three drugs, and therefore it was used for comparing their accumulation in several rat tissue cells after a single i.v. injection of each drug. In the kidney, immunoreactivity for each drug was distributed in essentially the same pattern and in the same strength 2 h after injection, but quite differently distributed in kidney cells thereafter, so that at 120-h post-injection significant amounts of DX and ER remained, but DR had almost completely vanished. Similar patterns of accumulation were observed in cells of other tissues including the pancreas, hair follicle, and stomach, with the exception of the intestine in which none of the three drugs remained after 120 h. These results appear to be supported by previous pharmacokinetic studies on the anthracyclines. The mechanism for such differences among the three drugs remains obscure, but the hydroxyl group at C-14 of DX and ER molecule might be related to the strong propensity of DX and ER to accumulate in tissue cells. The present results should contribute to the understanding of the mechanisms of the differences in the pharmacokinetics, as well as the possibly in anti-tumor activities of the anthracyclines.     

Identification and functional characterization of liposome tubulation protein from magnetotactic bacteria

Magnetotactic bacteria synthesize intracellular magnetosomes that are comprised of membrane‐enveloped magnetic crystals. In this study, to identify the early stages of magnetosome formation, we isolated magnetosomes containing small magnetite crystals and those containing regular‐sized magnetite crystals from Magnetospirillum magneticum AMB‐1. This was achieved by using a novel size fractionation technique, resulting in the identification of a characteristic protein (Amb1018/MamY) from the small magnetite crystal fraction. The gene encoding MamY was located in the magnetosome island. Like the previously reported membrane deformation proteins, such as bin/amphiphysin/Rvs (BAR) and the dynamin family proteins, recombinant MamY protein bound directly to the liposomes, causing them to form long tubules. We established a mamY gene deletion mutant (ΔmamY) and analysed MamY protein localization in it for functional characterization of the protein in vivo. The ΔmamY mutant was found to have expanded magnetosome vesicles and a greater number of small magnetite crystals relative to the wild‐type strain, suggesting that the function of the MamY protein is to constrict the magnetosome membrane during magnetosome vesicle formation, following which, the magnetite crystals grow to maturity within them.     

Comparative characterization of pulmonary surfactant aggregates and alkaline phosphatase isozymes in human lung carcinoma tissue

Alkaline phosphatase (AP) isozymes are surfactant-associated proteins (SPs). Since several different AP isozymes have been detected in the pneumocytes of lung cancer patients, we attempted to identify the relationship between pulmonary surfactant aggregate subtypes and AP isozymes. Pulmonary surfactant aggregates were isolated from carcinoma and non-carcinoma tissues of patients with non-small cell carcinoma of the lung. Upon analysis, ultraheavy, heavy, and light surfactant aggregates were detected in the non-carcinoma tissues, but no ultraheavy surfactant aggregates were found in the carcinoma tissues. Surfactant-associated protein A (SP-A) was detected as two bands (a 27-kDa band and a 54-kDa band) in the ultraheavy, heavy, and light surfactant aggregates found in the non-carcinoma tissues. Although both SP-A bands were detected in the heavy and light surfactant aggregates from adenocarcinoma tissues, the 54-kDa band was not detected in squamous cell carcinoma tissues. Liver AP (LAP) was detected in the heavy and light surfactant aggregates from both non-carcinoma and squamous carcinoma tissues, but not in heavy surfactant aggregates from adenocarcinoma tissues. A larger amount of bone type AP (BAP) was found in light surfactant aggregate fractions from squamous cell carcinomas than those from adenocarcinoma tissues or non-carcinoma tissues from patients with either type of cancer. LAP, BAP, and SP-A were identified immunohistochemically in type II pneumocytes from non-carcinoma tissues and adenocarcinoma cells, but no distinct SP-A staining was observed in squamous cell carcinoma tissues. The present study has thus revealed several differences in pulmonary surfactant aggregates and AP isozymes between adenocarcinoma tissue and squamous cell carcinoma tissue.     

High-throughput SNP detection using nano-scale engineered biomagnetite

A semi-automated system for the large-scale detection of single nucleotide polymorphisms (SNPs) has been developed based on allele-specific oligonucleotide hybridization and thermal dissociation curve analysis using nano-scale engineered biomagnetite (bacterial magnetic particles; BacMPs). For reliable detection in large numbers of samples, several conditions for the capture of target DNA on nano-sized BacMPs and the denaturation of double-stranded DNA were optimized. The most efficient target DNA capture was observed using short PCR amplicons (69 bp). Captured DNAs were denatured using 50 mM NaOH. With these optimizations, large-scale SNP detection was performed on 822 samples of the transforming growth factor (TGF)-beta1 gene, which is rich in both GC content and repetitive sequences. High reliability for the semi-automated BacMP-based SNP detection system was confirmed following comparison to traditional sequencing-based methods.     

Detection of epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) using a fully automated system with a nano-scale engineered biomagnetite

A fully automated system using nano-scale engineered biomagnetite was developed to detect mutations in the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer (NSCLC). Bacterial magnetic particles (BacMPs) were isolated from the magnetic bacterium Magnetospirillum magneticum AMB-1 and conjugated to streptavidin. Biotin-labeled target PCR products were then captured with the BacMPs, hybridized with the detection probe and detected by fluorescence signaling. The process was performed using a newly designed automated processor equipped with an XYZ mobile arm containing a 96-way automated pipetter, reagent dispenser and fluorescence detector. Two types of somatic mutations (in-frame deletions and point substitutions) in the EGFR gene were successfully identified within 3.5h using this system, suggesting that this system could be used in clinical tests of EGFR gene mutations in lung cancer, and potentially other cancer, patients. Additionally, a very low mutation rate could be detected in these samples.     

Spirodiketopiperazine-based CCR5 antagonists: Lead optimization from biologically active metabolite

Hydroxylated derivatives were designed and synthesized based on the information of oxidative metabolites. Compounds derived from beta-substituted (2R,3R)-2-amino-3-hydroxypropionic acid showed improved inhibitory activities against the binding of MIP-1alpha to human CCR5, compared with the non-hydroxylated derivatives and the other isomers.     

Poecillastrin D: a new cytotoxin of the chondropsin class from marine sponge Jaspis serpentina

Poecillastrin D (2) was isolated together with poecillastrin C (1) from the deep sea sponge, Japsis serpentina. Its structure was elucidated to be that of a macrolide lactam by spectroscopic methods. These compounds showed potent cytotoxicity against various tumor cell lines.