Identification of chlorophyllide a oxygenase in the Prochlorococcus genome by a comparative genomic approach

Chl b is a major photosynthetic pigment of peripheral antenna complexes in chlorophytes and prochlorophytes. Chl b is synthesized by chlorophyllide a oxygenase (CAO), an enzyme that has been identified from higher plants, moss, green algae and two groups of prochlorophytes, Prochlorothrix and Prochloron. Based on these results, we previously proposed the hypothesis that all of the Chl b synthesis genes have a common origin. However, the CAO gene is not found in whole genome sequences of Prochlorococcus although a gene which is distantly related to CAO was reported. If Prochlorococcus employs a different enzyme, a Chl synthesis gene should have evolved several times on the different phylogenetic lineages of Prochlorococcus and other Chl b-containing organisms. To examine these hypotheses, we identified a Prochlorococcus Chl b synthesis gene by using a combination of bioinformatics and molecular genetics techniques. We first identified Prochlorococcus-specific genes by comparing the whole genome sequences of Prochlorococcus marinus MED4, MIT9313 and SS120 with Synechococcus sp. WH8102. Synechococcus is closely related to Prochlorococcus phylogenetically, but it does not contain a Chl b synthesis gene. By examining the sequences of Prochlorococcus-specific genes, we found a candidate for the Chl b synthesis gene and introduced it into Synechocystis sp. PCC6803. The transformant cells accumulated Chl b, indicating that the gene product catalyzes Chl b synthesis. In this study, we discuss the evolution of CAO based upon the molecular phylogenetic studies we performed.     

Chromosome-shuffling technique for selected chromosomal segments in Saccharomyces cerevisiae

We describe a novel chromosome engineering technique for shuffling selected regions of chromosomes from two strains in Saccharomyces cerevisiae: The technique starts with the construction of MAT a and MATα strains in which a particular chromosome is split at exactly the same site in both strains such that the split chromosomes generated are marked with different markers. The two strains are then crossed, and the resultant diploid is cultivated in nutrient medium to induce loss of the split chromosome originating from either of the strains. We predicted that some of these clones that are hemizygous for the split chromosome would spontaneously restore a homozygous configuration of the split chromosome during cultivation. We verified this prediction by tetrad analysis and quantitative Southern hybridization analysis, indicating that it is possible to create diploid hybrids in which a selected region of a chromosome from one strain is replaced by the corresponding chromosomal region from another strain. We also found that some chromosomal segments maintain a hemizygous state. This novel technique, which we call ‘chromosome shuffling’, could provide a new tool to analyze phenotypic alterations caused by the replacement or hemizygosity of a selected chromosomal region in not only laboratory but also industrial strains of S. cerevisiae.     

Structure-activity studies on 1,3-dioxane-2-carboxylic acid derivatives, a novel class of subtype-selective peroxisome proliferator-activated receptor alpha (PPARalpha) agonists

A series of 1,3-dioxane carboxylic acid derivatives was synthesized and evaluated for human PPAR transactivation activity. Structure-activity relationships on the phenyloxazole moiety of the lead compound 3 revealed that the introduction of small hydrophobic substituents at the 4-position of the terminal phenyl ring increased the PPARalpha agonist activity, and that the oxazole heterocycle was essential to the maintenance of both potency and PPARalpha subtype-selectivity. This investigation led to the identification of 14d (NS-220) and 14i as highly potent and selective human PPARalpha agonists. In KK-A(y) type 2 diabetic mice, these compounds significantly lowered plasma triglyceride and very-low-density plus low-density lipoprotein cholesterol levels while simultaneously raising HDL cholesterol levels. Our results suggest that highly potent and subtype-selective PPARalpha agonists will be promising drugs for the treatment of metabolic disorders in type 2 diabetes.     

Perylene-conjugated pyrrole polyamide as a sequence-specific fluorescent probe

Perylene-conjugated pyrrole (Py)-polyamide 2 was designed and synthesized using the Fmoc solid-phase synthesis and a subsequent Sonogashira coupling reaction with 3-bromoperylene. Interestingly, conjugate 2 did not luminesce in water at 313 nm irradiation but was turned on in the presence of target double-stranded (ds) DNA, and showed strong emission with increasing DNA concentration, in particularly, by the binding to the target telomere sequences through heterodimer formation with partner 3. Importantly, the excitation spectrum of 2 clearly indicates that the Py and Imidazole (Im) moieties in the polyamide effectively sensitize the perylene moiety to give rise to fluorescence emission. Energy transfer would occur from the Py moiety to the perylene. Thus, screening of perylene-conjugates will allow us to develop a novel "molecular light switch" with sequence-specificity.     

Detection of triplet repeat sequences in the double-stranded DNA using pyrene-functionalized pyrrole-imidazole polyamides with rigid linkers

Methods for sequence-specific detection in double-stranded DNA (dsDNA) are becoming increasingly useful and important as diagnostic and imaging tools. Recently, we designed and synthesized pyrrole (Py)-imidazole (Im) polyamides possessing two pyrene moieties, 1, which showed an increased excimer emission in the presence of (CAG)(12)-containing oligodeoxynucleotides (ODN) 1 and 2. In this study, we synthesized bis-pyrenyl Py-Im polyamides with rigid linkers 2, 3, and 4 to improve their fluorescence properties. Among the conjugates, 2 showed a marked increase in excimer emission, which was dependent on the concentration of the target ODN and the number of CAG repeats in the dsDNA. Unlike conjugate 1, which has flexible linkers, the excimer emission intensity of 2 was retained at over 85%, even after 4h. Py-Im polyamides have the potential to be important diagnostic molecules for detecting genetic differences between individuals.     

The Y chromosome-specific STS marker MS2 and its peripheral regions on the Y chromosome of the dioecious plant Silene latifolia.

Sex determination in Silene latifolia uses the XX/XY system. The recent evolution of dioecy in S. latifolia provides a unique opportunity to study the early stages of Y chromosome evolution. However, the current Y chromosome map still contains many large gaps with no available markers. In this study, a sequence tagged site (STS) marker, MS2, was isolated and mapped to the same locus as L8 on the Y chromosome. To investigate the peripheral regions of MS2, a bacterial artificial chromosome (BAC) library was constructed from a male plant, and the BAC clone containing MS2 (MS2-9d12F) was isolated from 32 640 clones with an average insert size of 115 kb. A 109-kb insert of the BAC clone was analyzed. BLASTX analysis showed 11 sequences similar to some known proteins, most of which are retrotransposon-like elements. The ORF Finder predicted 9 ORFs within MS2-9d12F. RT-PCR analyses revealed that only 4 of the 9 predicted ORFs are expressed in both male and female plants. These 4 ORFs are candidates for genes having counterparts on both the X and Y chromosomes. Dot-matrix plot analysis and a BLASTN search revealed LTR-like sequences close to the retrotransposon-like elements and high similarity to 3 known genomic sequences of S. latifolia. These results suggest an accumulation of retrotransposons and segmental duplications in peripheral regions of MS2 during the early stage of sex chromosome evolution.     

Expression of EGFP-spider dragline silk fusion protein in BmN cells and larvae of silkworm showed the solubility is primary limit for dragline proteins yield

Spider dragline silk is a unique fibrous protein with combination of tensile strength and elasticity, but the isolation of large amount of silk from spiders is not feasible. In this paper, we used a newly established Bac-to-Bac/BmNPV Baculovirus expression system to express the recombinant spider (Nephila clavata) dragline silk protein (MaSp1) fused EGFP in BmN cells and larvae of silkworm. A 70 kDa fusion protein was visualized after rBacmid/BmNPV/drag infection by SDS-PAGE and immunoblotting analysis. Fusion protein expressed in the BmN cells probably occupied five percent of the cell total protein; In a silkworm larva, approximately 6 mg fusion proteins were expressed. Solubility analysis of the expressed spider dragline silk protein indicated that 60% fusion protein is insoluble. EGFP fluorescence showed that fusion protein is tend to form aggregate by self assemblage. The results indicated the solubility is the primary limit for spider dragline proteins yield. It also suggested that directly produce fibrous spider silk in the secreting-silk organs of the transgenic silkworm larvae might be a better method.     

Induction of apoptosis by cigarette smoke via ROS-dependent endoplasmic reticulum stress and CCAAT/enhancer-binding protein-homologous protein (CHOP)

In this report, we investigated a role of endoplasmic reticulum (ER) stress in cigarette smoke (CS)-induced apoptosis of human bronchial epithelial cells (hBEC). Exposure of hBEC to CS or CS extract (CSE) caused expression of endogenous ER stress markers GRP78 and CHOP and induction of apoptosis evidenced by nuclear condensation, membrane blebbing, and activation of caspase-3 and caspase-4. In vivo exposure of mice to CS also caused induction of GRP78 and CHOP in the lung. Attenuation of ER stress by overexpression of ER chaperone GRP78 or ORP150 significantly attenuated CSE-triggered apoptosis. Exposure of hBEC to CSE caused generation of reactive oxygen species, and treatment with antioxidants inhibited CSE-induced apoptosis. Interestingly, antioxidants including a scavenger of O(2)(*-) blunted induction of CHOP by CSE without affecting the level of GRP78, and dominant-negative inhibition of CHOP abolished CSE-induced apoptosis. Furthermore, a generator of O(2)(*-) selectively induced CHOP and apoptosis in hBEC. Our results revealed that: (1) CS induces ER stress in vitro and in vivo, (2) ER stress mediates CS-triggered apoptosis downstream of oxidative stress, (3) CS-initiated apoptosis is caused through oxidative stress-dependent induction of CHOP, (4) O(2)(*-) may play a dominant role in this process, and (5) oxidative stress-independent induction of GRP78 counterbalances the proapoptotic action of CHOP.     

Removal of linear and branched alkylphenols from aqueous solutions with horseradish peroxidase

Alkylphenols were effectively treated with horseradish peroxidase at pH 7.0 and 30 degrees C in the presence of H(2)O(2) and poly(ethylene glycol) irrespective of the relative position or isomeric form of the alkyl chains. Water-insoluble oligomer precipitates were readily filtered out after enzymatic treatment, and transparent and colorless solutions were obtained for all p- and m-alkylphenols used.     

Synthesis of D-trigalacturonic acid methylglycoside and conformational comparison with its sulfur analogue

D-Trigalacturonic acid methylglycoside (3) was synthesized to evaluate the previously synthesized sulfur analogue 1 by comparison. The NOE experiments revealed that both 3 and 1 took on a similar conformation around their glycosyl linkage.