Rapid and effective detection of anthrax spores in soil by PCR

AIMS: To detect Bacillus anthracis DNA from soil using rapid and simple procedures. METHODS AND RESULTS: Various amounts of B. anthracis Pasteur II spores were added artificially to 1 g of soil, which was then washed with ethanol and sterile water. Enrichment of the samples in trypticase soy broth was performed twice. A DNA template was prepared from the second enrichment culture using a FastPrep instrument. The template was then used for nested and real-time polymerase chain reaction (PCR) with B. anthracis-specific primers, to confirm the presence of B. anthracis chromosomal DNA and the pXO1/pXO2 plasmids. CONCLUSIONS: One cell of B. anthracis in 1 g of soil could be detected by nested and real-time PCR. The usefulness of the PCR method using field samples was also confirmed. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that this could be a useful method for detecting anthrax-spore contaminated soil with high sensitivity. Its application could have great impact on the progress of epidemiological surveillance.     

Expression of a germination-specific amidase, SleB, of Bacilli in the forespore compartment of sporulating cells and its localization on the exterior side of the cortex in dormant spores

A germination-specific amidase of bacilli is a major spore-lytic enzyme that is synthesized with a putative signal sequence and hydrolyses spore cortex in situ. The sleB gene encoding this amidase in Bacillus subtilis and Bacillus cereus was expressed in the forespore compartment of sporulating cells under the control of sigmaG, as shown by Northern blot and primer extension analyses. The forespore-specific expression of B. subtilis sleB was further indicated by the forespore-specific accumulation of a SleB-green fluorescent protein fusion protein from which a putative secretion signal of SleB was deleted. Immunoelectron microscopy with anti-SleB antiserum and a colloidal gold-immunoglobulin G complex showed that the enzymes from both Bacillus species are located just inside the spore coat layer in the dormant spore, and in the dormant spore, the amidases appear exist in a mature form lacking a signal sequence. These results indicate that SleB is translocated across the forespore's inner membrane by a secretion signal peptide and is deposited in cortex layer synthesized between the forespore inner and outer membranes. The peripheral location of the spore-lytic enzymes in the dormant spore suggests that spore germination is initiated at the exterior of the cortex.     

Structure and host recognition of serotype 13 glycopeptidolipid from Mycobacterium intracellulare

The Mycobacterium avium-M. intracellulare complex (MAIC) is divided into 28 serotypes by a species-specific glycopeptidolipid (GPL). Previously, we clarified the structures of serotype 7 GPL and two methyltransferase genes (orfA and orfB) in serotype 12 GPL. This study elucidated the chemical structure, biosynthesis gene, and host innate immune response of serotype 13 GPL. The oligosaccharide (OSE) structure of serotype 13 GPL was determined to be 4-2'-hydroxypropanoyl-amido-4,6-dideoxy-β-hexose-(1 → 3)-4-O-methyl-α-L-rhamnose-(1 → 3)-α-L-rhamnose-(1 → 3)-α-L-rhamnose-(1 → 2)-α-L-6-deoxy-talose by using chromatography, mass spectrometry, and nuclear magnetic resonance (NMR) analyses. The structure of the serotype 13 GPL was different from those of serotype 7 and 12 GPLs only in O-methylations. We found a relationship between the structure and biosynthesis gene cluster. M. intracellulare serotypes 12 and 13 have a 1.95-kb orfA-orfB gene responsible for 3-O-methylation at the terminal hexose, orfB, and 4-O-methylation at the rhamnose next to the terminal hexose, orfA. The serotype 13 orfB had a nonfunctional one-base missense mutation that modifies serotype 12 GPL to serotype 13 GPL. Moreover, the native serotype 13 GPL was multiacetylated and recognized via Toll-like receptor 2. The findings presented here imply that serotypes 7, 12, and 13 are phylogenetically related and confirm that acetylation of the GPL is necessary for host recognition. This study will promote better understanding of the structure-function relationships of GPLs and may open a new avenue for the prevention of MAIC infections.     

Human aquaporin adipose (AQPap) gene. Genomic structure, promoter analysis and functional mutation.

Aquaporin adipose (AQPap), which we identified from human adipose tissue, is a glycerol channel in adipocyte [Kishida et al. (2000) J. Biol. Chem. 275, 20896-20902]. In the current study, we determined the genomic structure of the human AQPap gene, and identified three AQPap-like genes that resembled (approximately 95%) AQPap, with little expression in human tissues. The AQPap promoter contained a putative peroxisome proliferator response element (PPRE) at -46 to -62, and a putative insulin response element (IRE) at -542/-536. Deletion of the PPRE abolished the pioglitazone-mediated induction of AQPap promoter activity in 3T3-L1 adipocytes. Deletion and single base pair substitution analysis of the IRE abolished the insulin-mediated suppression of the human AQPap gene. Analysis of AQPap sequence in human subjects revealed three missense mutations (R12C, V59L and G264V), and two silent mutations (A103A and G250G). The cRNA injection of the missense mutants into Xenopus oocytes revealed the absence of the activity to transport glycerol and water in the AQPap-G264V protein. In the subject homozygous for AQPap-G264V, exercise-induced increase in plasma glycerol was not observed in spite of the increased plasma noradrenaline. We suggest that AQPap is responsible for the increase of plasma glycerol during exercise in humans.     

Saturated and trans-unsaturated fatty acids elicit high levels of superoxide generation in intact and cell-free preparations of neutrophils

Saturated and trans-unsaturated fatty acids, such as laurate and elaidate, elicited O2- generation in intact porcine and human neutrophils and also in a cell-free preparation of porcine neutrophils. The activities thus induced were comparable to those induced by cis-unsaturated fatty acids. However, the activation by saturated or trans-unsaturated fatty acids was depressed almost completely in the presence of Ca2+ at around 1 mM, which is usually contained in the media for phagocytes. In contrast, the activation by cis-unsaturated fatty acids such as arachidonate was scarcely affected by Ca2+. These findings appear to demand reevaluation of the effects of long chain fatty acids on the respiratory burst system in phagocytes.     

Structure-based design of a leucine zipper protein with new DNA contacting region

We have employed a structure-based design to construct a small folding domain from the F-actin bundling protein villin that contains the amino acids necessary for the DNA binding of the basic leucine zipper protein GCN4 and have compared its DNA binding with GCN4. The monomeric motif folds into a stable domain and binds DNA in a rigid-body mechanism, while its affinity is not higher than that of the basic region peptide. The addition of the leucine zipper region to the folded domain restored its sequence-specific DNA binding comparable to that of GCN4. Unlike the monomeric folded domain, its leucine zipper derivative undergoes a conformational change upon DNA binding. CD spectral and thermodynamic studies indicate that the DNA-contacting region is folded in the presence or absence of DNA and suggest that the junction between the DNA-contacting and the leucine zipper regions transits to a helix in the presence of DNA. These results demonstrate that the structural transition outside the direct-contacting region, which adjusts the precise location of the DNA-contacting region, plays a critical role in the specific complex formation of basic leucine zipper proteins.     

'Working' cardiomyocytes exhibiting plateau action potentials from human placenta-derived extraembryonic mesodermal cells

The clinical application of cell transplantation for severe heart failure is a promising strategy to improve impaired cardiac function. Recently, an array of cell types, including bone marrow cells, endothelial progenitors, mesenchymal stem cells, resident cardiac stem cells, and embryonic stem cells, have become important candidates for cell sources for cardiac repair. In the present study, we focused on the placenta as a cell source. Cells from the chorionic plate in the fetal portion of the human placenta were obtained after delivery by the primary culture method, and the cells generated in this study had the Y sex chromosome, indicating that the cells were derived from the fetus. The cells potentially expressed 'working' cardiomyocyte-specific genes such as cardiac myosin heavy chain 7beta, atrial myosin light chain, cardiac alpha-actin by gene chip analysis, and Csx/Nkx2.5, GATA4 by RT-PCR, cardiac troponin-I and connexin 43 by immunohistochemistry. These cells were able to differentiate into cardiomyocytes. Cardiac troponin-I and connexin 43 displayed a discontinuous pattern of localization at intercellular contact sites after cardiomyogenic differentiation, suggesting that the chorionic mesoderm contained a large number of cells with cardiomyogenic potential. The cells began spontaneously beating 3 days after co-cultivation with murine fetal cardiomyocytes and the frequency of beating cells reached a maximum on day 10. The contraction of the cardiomyocytes was rhythmical and synchronous, suggesting the presence of electrical communication between the cells. Placenta-derived human fetal cells may be useful for patients who cannot supply bone marrow cells but want to receive stem cell-based cardiac therapy.     

Oxygenation-linked changes in the ultraviolet absorption and circular dichroism spectra of spiny lobster hemocyanin

As an approach to elucidate the mechanism of the protein structure change in the cooperative ligand binding, the UV difference and CD spectra of aromatic residues in Panulirus japonicus (spiny lobster) hemocyanin were examined. The native hemocyanin showed an O2-induced narrow-banded change in the absorption spectrum around 290 nm, which was not affected by pH in the range of 7.5 to 9.5. When the native hexameric protein was stripped of divalent cations with EDTA (at pH 7.5), the magnitude of the narrow-banded difference was reduced to about half, whereas it was almost completely abolished on dissociation into subunits (stripped at pH 9.5). The magnitude of the absorption change was found to be proportional to the degree of O2 saturation in the native and stripped hemocyanins. It was inferred that the spectral difference reflects a tertiary structure change directly linked to the oxygenation, though it depends greatly on the subunit association. Panulirus hemocyanin showed negative CD bands in the region of 260 to 300 nm, the intensities of which were considerably reduced by oxygenation and also by dissociation into subunits.