The cls gene of Escherichia coli is responsible for the synthesis of a major membrane phospholipid, cardiolipin, and has been proposed to encode cardiolipin synthase. This gene cloned on a pBR322 derivative was disrupted by either insertion of or replacement with a kanamycin-resistant gene followed by exchange with the homologous chromosomal region. The proper genomic disruptions were confirmed by Southern blot hybridization and a transductional linkage analysis. Both types of disruptants had essentially the same properties; cardiolipin synthase activity was not detectable, but the strains grew well, although their growth rates and final culture densities were lower than those of the corresponding wild-type strains and strains with the classical cls-l mutation. A disruptant harboring a plasmid that carried the intact cls gene grew normally.The results indicate that the cls gene and probably the cardiolipin synthase are dispensable for E. coli but may confer growth or survival advantages. Low but definite levels of cardiolipin were synthesized by all the disruptants. Cardiolipin content of the cis mutants depended on the dosage of the pss gene,-and attempts to transfer a null allele of the cls gene into a pss-l mutant were unsuccessful. We point out the possibilities of minor cardiolipin formation by phosphatidylserine synthase and of the essential nature of cardiolipin for the survival of E. coli cells.Cardiolipin, one of the major phospholipids of Escherichia coli, is unique in its structure among membrane lipids (i.e., tetraacyl structure with two phosphate groups) and has been postulated to play specific roles in membrane functions. It is synthesized from two molecules of phosphatidylglycerol by cardiolipin synthase (4, 20), which differs from its eucaryotic counterpart that utilizes CDP-diacylglycerol and phosphatidylglycerol as substrates (21). The gene cls is responsible for cardiolipin formation, and several lines of evidence indicate, though not definitely, that it is the structural gene for cardiolipin synthase (10).An E. coli mutation (cls-J) that results in a defective cardiolipin synthase has been isolated (14), and the cells with this mutation have been shown to display only minor growth phenotypes, despite decreased levels of cardiolipin (14, 18). Therefore, it has been uncertain whether the cls gene and cardiolipin are essential in E. coli cells or whether the residual low levels of cardiolipin observed maintain membrane functions that are dependent on this particular phospholipid.To determine whether the cls gene is essential and to analyze the physiological roles, if any, of this gene, an attempt to obtain a null mutation of this gene seemed most useful. This paper describes the construction, by genetic manipulations, of such mutants and their characteristic properties. The results show that the cls gene is not essential. Cardiolipin was formed at low levels even in the null cls mutants. We also present results that favor a hypothesis of secondary cardiolipin formation by phosphatidylserine synt...
An acinar morphogenesis inhibitor named fusarisetin A (1) that possesses both an unprecedented carbon skeleton and a new pentacyclic ring system has been identified from an in-house fractionated fungal library using a three-dimensional matrigel-induced acinar morphogenesis assay system. The structure of 1 was determined in detail by NMR and circular dichroism spectroscopy, X-ray analysis, and chemical reaction experiments.
Seven new compounds (aflaquinolones A – G; 1 – 7) containing dihydroquinolin-2-one and terpenoid units have been isolated from two different fungal sources. Two of these metabolites (1 and 2) were obtained from a Hawaiian fungicolous isolate of Aspergillus sp. (section Flavipedes; MYC-2048 = NRRL 58570), while the others were obtained from a marine Aspergillus isolate (SF-5044) collected in Korea. The structures of these compounds were determined mainly by analysis of NMR and MS data. Relative and absolute configurations were assigned on the basis of NOESY data and 1H NMR J-values, comparison of calculated and experimental ECD spectra, and analysis of a Mosher’s ester derivative of 2. Several known compounds, including alantrypinone, aspochalasins I and J, methyl-3,4,5-trimethoxy-2((2-((3-pyridinylcarbonyl)amino) benzoyl)amino) benzoate, and trans-dehydrocurvularin were also encountered in the extract of the Hawaiian isolate.
Fumitremorgin C, a diketopiperazine mycotoxin produced by Aspergillus fumigatus, is a potent and specific inhibitor of breast cancer resistance protein (BCRP). Elucidation of the fumitremorgin C biosynthetic pathway provides a strategy for new drug design. A structure-activity relationship study based on metabolites related to the ftm gene cluster revealed that the process most crucial for inhibitory activity against BCRP was cyclization to form fumitremorgin C. To determine the gene involved in the cyclization reaction, targeted gene inactivation was performed with candidate genes in the ftm cluster. Analysis of the gene disruptants allowed us to identify ftmE, one of the cytochrome P450 genes in the cluster, as the gene responsible for the key step in fumitremorgin biosynthesis. Additionally, we demonstrated that the other two cytochrome P450 genes, ftmC and ftmG, were involved in hydroxylation of the indole ring and successive hydroxylation of fumitremorgin C, respectively.
Two new oxepin-containing (1 and 2) and two diketopiperazine-type alkaloids (3 and 4) have been isolated from an EtOAc extract of the marine-derived fungus Aspergillus sp. SF-5044. The structures of these metabolites were determined through analysis of NMR and MS data, along with Marfey's method. Compound 1 showed weak growth inhibitory activity against a small panel of cell lines.
Although the "gold standard" for diagnosis of tuberculous meningitis (TBM) is bacterial isolation of Mycobacterium tuberculosis, there are still several complex issues. Recently, we developed an internally controlled novel wide-range quantitative nested real-time PCR (WR-QNRT-PCR) assay for M. tuberculosis DNA in order to rapidly diagnose TBM. For use as an internal control calibrator to measure the copy number of M. tuberculosis DNA, an original new-mutation plasmid (NM-plasmid) was developed. Due to the development of the NM-plasmid, the WR-QNRT-PCR assay demonstrated statistically significant accuracy over a wide detection range (1 to 10 5 copies). In clinical applications, the WR-QNRT-PCR assay revealed sufficiently high sensitivity (95.8%) and specificity (100%) for 24 clinically suspected TBM patients. In conditional logistic regression analysis, a copy number of M. tuberculosis DNA (per 1 ml of cerebrospinal fluid) of >8,000 was an independent risk factor for poor prognosis for TBM (i.e., death) (odds ratio, 16.142; 95% confidence interval, 1.191 to 218.79; P value, 0.0365). In addition, the copy numbers demonstrated by analysis of variance statistically significant alterations (P < 0.01) during the clinical treatment course for 10 suspected TBM patients. In simple regression analysis, the significant correlation (R 2 ؍ 0.597; P < 0.0001) was demonstrated between copy number and clinical stage of TBM. We consider the WR-QNRT-PCR assay to be a useful and advanced assay technique for assessing the clinical treatment course of TBM.Tuberculous meningitis (TBM) is the severest form of infection of Mycobacterium tuberculosis, causing death or severe neurological defects in more than half of those affected in spite of antituberculosis treatment (ATT) (1,2,8,18). The diagnosis of TBM remains a complex issue, because the most widely used conventional bacteriological detection methods, such as direct smear for acid-fast bacilli (AFB) and culture for M. tuberculosis, are unable to rapidly detect M. tuberculosis with sufficient sensitivity in the acute phase of TBM (3-13, 18, 19). In 2006, we designed a novel internally controlled quantitative nested real-time PCR (QNRT-PCR) assay based on TaqMan PCR (Applied Biosystems) (15). Moreover, based on this original QNRT-PCR (OR-QNRT-PCR) assay, an improved wide-range QNRT-PCR (WR-QNRT-PCR) assay was developed (17). For use as a "calibrator" in WR-QNRT-PCR assay, a new internal control was constructed (17).In the preliminary experiments, the WR-QNRT-PCR assay demonstrated significantly improved quantitative accuracy and had a wide detection range (1 to 10 5 copies) compared to what was seen for the OR-QNRT-PCR assay (17).In this study, we tried to quantitatively detect M. tuberculosis DNA in actual cerebrospinal fluid (CSF) samples by using the WR-QNRT-PCR assay. In addition, the clinical usefulness of this novel assay technique for the rapid and accurate diagnosis of TBM and for assessing the clinical course of TBM was examined. MATERIALS AND METHODSThis study was approv...
Matrix metalloproteinase (MMP)-9, the 92-kDa type IV collagenase, contributes to tumor invasion and metastases, and strategies to down-regulate its expression could ultimately be of clinical utility. A pyrrole-imidazole (PI) polyamide that targets the activator protein-1 (AP-1)-binding site of the MMP-9 promoter was designed and synthesized as a gene-silencing agent for tumor metastases. The synthesized product showed selective DNA binding ability. The MMP-9 PI polyamide significantly inhibited MMP-9's mRNA expression, protein level, and enzymatic activity in human breast adenocarcinoma cells (MDA-MB-231). Furthermore, the MMP-9 PI polyamide inhibited migration and invasion by in vitro wound-healing and matrigel-invasion assay. The FITC-labeled PI polyamide was localized in nuclei in 45 min of incubation with an MDA-MB-231 cell and remained in the nuclei for up to 96 h after incubation in vitro. It was also quickly localized in the mouse cellular nuclei of many tissues, including liver, kidney, and spleen, after intravenous injection without using any drug-delivery system. Moreover, the polyamide treatment significantly decreased metastasis in a mouse model of liver metastasis. Our results suggest that this PI polyamide, which targets the MMP-9 gene promoter, can be a novel MMP-9 down-regulating molecule for antimetastasis.
[structure: see text] Azaspirene isolated from the fungus Neosartorya sp. is a novel angiogenesis inhibitor with a 1-oxa-7-azaspiro[4.4]non-2-ene-4,6-dione skeleton. Azaspirene inhibits the endothelial migration induced by vascular endothelial growth factor (ED100 = 27.1 microM).
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