Horizontal gene transfer (HGT) as a mechanism of disseminating RDX-degrading activity among Actinomycete bacteria.

AIMS: Hexahydro-1,3,5-trinitro-1,3,5,-triazine (RDX) is a cyclic nitramine explosive that is a major component in many high-explosive formulations and has been found as a contaminant of soil and groundwater. The RDX-degrading gene locus xplAB, located on pGKT2 in Gordonia sp. KTR9, is highly conserved among isolates from disparate geographical locations suggesting a horizontal gene transfer (HGT) event. It was our goal to determine whether Gordonia sp. KTR9 is capable of transferring pGKT2 and the associated RDX degradation ability to other bacteria. METHODS AND RESULTS: We demonstrate the successful conjugal transfer of pGKT2 from Gordonia sp. KTR9 to Gordonia polyisoprenivorans, Rhodococcus jostii RHA1 and Nocardia sp. TW2. Through growth and RDX degradation studies, it was demonstrated that pGKT2 conferred to transconjugants the ability to degrade and utilize RDX as a nitrogen source. The inhibitory effect of exogenous inorganic nitrogen sources on RDX degradation in transconjugant strains was found to be strain specific. CONCLUSIONS: Plasmid pGKT2 can be transferred by conjugation, along with the ability to degrade RDX, to related bacteria, providing evidence of at least one mechanism for the dissemination and persistence of xplAB in the environment. SIGNIFICANCE AND IMPACT OF STUDY: These results provide evidence of one mechanism for the environmental dissemination of xplAB and provide a framework for future field relevant bioremediation practices.

Acetylation of fluoroquinolone antimicrobial agents by an Escherichia coli strain isolated from a municipal wastewater treatment plant.

AIMS: To isolate environmental bacteria capable of transforming fluoroquinolones to inactive molecules. METHODS AND RESULTS: Bacteria were isolated from the aerobic liquor of a wastewater treatment plant on a medium containing norfloxacin (100 mg l(-1)). Twenty-two isolates were highly resistant (minimal inhibitory concentration: 6.25-200 microg ml(-1)) to five fluoroquinolones and six of them were positive by PCR amplification for the aminoglycoside resistance gene aac(6')-Ib. Of these, only Escherichia coli strain LR09 had the ciprofloxacin-acetylating variant gene aac(6')-Ib-cr; HPLC and mass spectrometry showed that this strain transformed both ciprofloxacin and norfloxacin by N-acetylation. This bacterium also had mutations in the quinolone-resistance determining regions of the gyrA and parC genes. CONCLUSIONS: An E. coli isolate from wastewater, which possessed at least two distinct fluoroquinolone resistance mechanisms, inactivated ciprofloxacin and norfloxacin by N-acetylation. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of N-acetylation of fluoroquinolones by an aac(6')-Ib-cr-containing bacterium from an environmental source.

Two new monoterpene peroxide glycosides from Aster scaber.

The aerial part of Aster scaber Thunb. (Asteraceae) yielded two new monoterpene peroxide glycosides, (3S)-3-O-(3',4'-diangeloyl-beta-D-glucopyranosyloxy)-7-hydroperoxy-3,7-dimethylocta-1,5-diene (1) and (3S)-3-O-(3',4'-diangeloyl-beta-D-glucopyranosyloxy)-6-hydroperoxy-3,7-dimethylocta-1,7-diene (2), and five known compounds, alpha-spinasterol (3), germacra-4(15),5,10(14)-triene-1-beta-ol (4), 7-methoxy-4(15)-oppositen-1-beta-ol (5), 6alpha-methoxy-4(15)-eudesmane-1beta-ol (6) and alpha-spinasterol 3-O-beta-D-glucopyranoside (7). The structures were established by chemical and spectroscopic methods.

A new caffeoyl quinic acid from aster scaber and its inhibitory activity against human immunodeficiency virus-1 (HIV-1) integrase.

The phytochemical study of the aerial parts of Aster scaber Thunb. (Asteraceae) yielded a new caffeoyl quinic acid, (-) 3,5-dicaffeoyl-muco-quinic acid (2) and three known compounds, (-) 3,5-dicaffeoyl quinic acid (1), (-) 4,5-dicaffeoyl quinic acid (3), (-) 5-caffeoyl quinic acid (4). The structures were established by high resolution spectroscopic methods. The antiviral effects against HIV-1 integrase of the compounds was evaluated. (-) 3,5-Dicaffeoyl-muco-quinic acid (2) exhibited potent antiviral activity with an IC50 value of 7.0 +/- 1.3 microg/ml.

[Proton-MR spectroscopy of the spinal bone marrow. An analysis of physiological signal behavior]. / Protonen-MR-Spektroskopie des Wirbelkörpermarks. Analyse des physiologischen Signalverhaltens.

BACKGROUND: Magnetic resonance imaging has shown to be a sensitive method for diagnostics of the red bone marrow, the composition of which changes physiologically and during pathological processes. However, the interpretation of MRI in patients with disorders of the red bone marrow is very difficult. The aim of this study was the characterization of the proton spectrum of healthy bone marrow and its age- and sex-dependent changes to obtain a data basis for measurements in patients. METHODS: 154 healthy volunteers have been examined. After imaging, a spectroscopic measurement was performed to determine the relative intensities of fat and water, and their respective T2 times. RESULTS: While T2 (water: 46.9 ms and fat: 75.4 ms) does not depend on age or sex, the relative signal intensity of fat increases by about 6% per decade. In the age groups between 31 and 50 years it diverges significantly between men (43.5%) and woman (32.5%) (p < or = 0.01, Mann-Whitney-Test. CONCLUSIONS: Proton spectroscopy can increase the reliability of diagnosis by offering information on composition of the marrow. The analysis of spectroscopic measurements requires exact knowledge about normal physiological values.

Inducible nitric oxide synthase inhibitors from Melia azedarach var. japonica.

In bioassay-guided search for inducible nitric oxide synthase (iNOS) inhibitory compounds from higher plants of South Korea, two beta-carboline alkaloids, 4-methoxy-1-vinyl-beta-carboline (1) and 4,8-dimethoxy-l-vinyl-beta-carboline (2) have been isolated from the cortex of Melia azedarach var. japonica. The structures of these compounds were elucidated on the basis of spectroscopic data. Compounds 1 and 2 showed marked inhibitory activity of iNOS on LPS- and interferon-gamma-stimulated RAW 264.7 cells.

Promoter upstream bent DNA activates the transcription of the Clostridium perfringens phospholipase C gene in a low temperature-dependent manner.

The phospholipase C gene (plc) of Clostridium perfringens possesses three phased A-tracts forming bent DNA upstream of the promoter. An in vitro transcription assay involving C.perfringens RNA polymerase (RNAP) showed that the phased A-tracts have a stimulatory effect on the plc promoter, and that the effect is proportional to the number of A-tracts, and more prominent at lower temperature. A gel retardation assay and hydroxyl radical footprinting revealed that the phased A-tracts facilitate the formation of the RNAP-plc promoter complex through extension of the contact region. The upstream (UP) element of the Escherichia coli rrnB P1 promoter stimulated the downstream promoter activity temperature independently, differing from the phased A-tracts. When the UP element was placed upstream of the plc promoter, low temperature-dependent stimulation was observed, although this effect was less prominent than that of the phased A-tracts. These results suggest that both the phased A-tracts and UP element cause low temperature-dependent activation of the plc promoter through a similar mechanism, and that the more efficient low temperature-dependent activation by the phased A-tracts may be due to an increase in the bending angle at a lower temperature.

Identification of metal ligands in the Clostridium histolyticum ColH collagenase.

A Clostridium histolyticum 116-kDa collagenase has an H415EXXH motif but not the third zinc ligand, as found in already characterized zinc metalloproteinases. To identify its catalytic site, we mutated the codons corresponding to the three conserved residues in the motif to other amino acid residues. The mutation affecting His415 or His419 abolished catalytic activity and zinc binding, while that affecting Glu416 did the former but not the latter. These results suggest that the motif forms the catalytic site. We also mutated the codons corresponding to other amino acid residues that are likely zinc ligands. The mutation affecting Glu447 decreased markedly both the enzymatic activity and the zinc content, while that affecting Glu446 or Glu451 had smaller effects on activity and zinc binding. These mutations caused a decrease in kcat but no significant change in Km. These results are consistent with the hypothesis that Glu447 is the third zinc ligand. The spacing of the three zinc ligands is the same in all known clostridial collagenases but not in other known gluzincins, indicating that they form a new gluzincin subfamily. The effects of mutations affecting Glu446 and Glu451 suggest that the two residues are also involved in catalysis, possibly through an interaction with the two zinc-binding histidine residues.

Gene duplication and multiplicity of collagenases in Clostridium histolyticum.

Clostridium histolyticum collagenase contains a number of different active components. Previously we have shown that colH encodes a 116-kDa collagenase (ColH) and a 98-kDa gelatinase. We purified a different 116-kDa collagenase (ColG) from the culture supernatant and sequenced its gene (colG). We also identified four other gelatinases (105, 82, 78, and 67 kDa) and determined their N-terminal amino acid sequences, all of which coincided with that of either ColG or ColH. Hybridization experiments showed that each gene is present in a single copy and each gene is transcribed into a single mRNA. These results suggest that all the gelatinases are produced from the respective full-length collagenase by the proteolytic removal of C-terminal fragments. The substrate specificities of the enzymes suggest that colG and colH encode class I and class II enzymes, respectively. Analysis of their DNA locations by pulsed-field gel electrophoresis and nucleotide sequencing of their surrounding regions revealed that the two genes are located in different sites on the chromosome. C. histolyticum colG is more similar to C. perfringens colA than to colH in terms of domain structure. Both colG and colA have a homologous gene, mscL, at their 3' ends. These results suggest that gene duplication and segment duplication have occurred in an ancestor cell common to C. histolyticum and C. perfringens and that further divergence of the parent gene produced colG and colA.

A study of the collagen-binding domain of a 116-kDa Clostridium histolyticum collagenase.

The Clostridium histolyticum 116-kDa collagenase consists of four segments, S1, S2a, S2b, and S3. A 98-kDa gelatinase, which can degrade denatured but not native collagen, lacks the C-terminal fragment containing a part of S2b and S3. In this paper we have investigated the function of the C-terminal segments using recombinant proteins. Full-length collagenase degraded both native type I collagen and a synthetic substrate, Pz-peptide, while an 88-kDa protein containing only S1 and S2a (S1S2a) degraded only Pz-peptide. Unlike the full-length enzyme, S1S2a did not bind to insoluble type I collagen. To determine the molecular determinant of collagen binding activity, various C-terminal regions were fused to the C terminus of glutathione S-transferase. S3 as well as S2bS3 conferred collagen binding. However, a glutathione S-transferase fusion protein with a region shorter than S3 exhibited reduced collagen binding activity. S3 liberated from the fusion protein also showed collagen binding activity, but not S2aS2b or S2b. S1 had 100% of the Pz-peptidase activity but only 5% of the collagenolytic activity of the full-length collagenase. These results indicate that S1 and S3 are the catalytic and binding domains, respectively, and that S2a and S2b form an interdomain structure.

Immunohistochemical analysis of late local skin reactions during rush venom immunotherapy.

During rush venom immunotherapy (VIT), about 65% of patients develop large local reactions (LLR) at the application site that last for at least 24 h. However, LLR subside during long-term treatment. To learn more about the provenance of infiltrating cells in late, local skin reactions during VIT, we analyzed the skin infiltrates of 23 Hymenoptera venom (HV)-allergic patients. Punch biopsies were obtained 24 h after s.c. injection of HV allergens from 23 HV-allergic patients and five nonallergic controls. Seven patients did not show LLR at the beginning of VIT. Ten patients had LLR when the dose of HV allergens was increased. Six patients showed reduced LLR after long-term treatment. Immunoenzymatic labeling of the cryostal sections with a panel of monoclonal antibodies was performed by the APAAP method. S.c. application of HV allergens induced a perivascular and periadnexial cutaneous mononuclear cell infiltrate consisting mainly of CD4+, CD45RO+; and HLA-DR+ cells in patients without clinically apparent LLR. In contrast, LLR were associated with a significant increase in total cells, CD4+ cells, CD8+ cells, CD11c+ cells, EG2+ cells, NP57+ cells, HLA-DR+ cells, CD45RO+ cells, CD45RA+ cells, CD23+ cells and CD25+ cells (P < 0.001). Decreased LLR after long-term VIT was correlated with a significantly reduced recruitment of CD4+ cells, EG2+ cells, and CD23+ cells as compared to LLR in the course of dose increases (P < 0.05), whereas the number of CD8+ cells, CD11c+ cells, NP57+ cells, and CD25+ cells remained high. Our data suggest that s.c. injections of HV allergens attract CD4+ helper T cells, of both the naive (CD45RA+) and memory (CD45RO+) phenotypes, to the allergen application site. LLR represent delayed allergic rather than toxic reactions to HV components and might be relevant to the development of clinical protection during VIT.

Expression of the colH gene encoding Clostridium histolyticum collagenase in Bacillus subtilis and its application to enzyme purification.

The colH gene encoding 116-kDa collagenase of Clostridium histolyticum (cColH) was cloned into an Escherichia coli-Bacillus subtilis shuttle vector to develop a method for purification of recombinant collagenase (rColH). When plasmid pJCM310 containing the colH gene was introduced into B. subtilis DB104 and the transformant was grown in LB broth at 37 C, stability of the plasmid was not maintained. However, stability was partly improved by growing the transformant in a modified LB broth containing 0.5 M sodium succinate with gentle shaking at 35 C. When the transformant was grown to an optical density of 0.4 at 600 nm in this medium, pJCM310 was stable and rColH was produced in sufficient amounts. rColH was purified to homogeneity by ammonium sulfate precipitation, gel filtration and ion-exchange chromatography. The yield of rColH from an 800-ml culture was 0.53 mg and its specific activity was estimated to be 1,210 U per mg of protein. The purified rColH was capable of degrading native type-I collagen fibril from bovine achilles tendon, as was demonstrated by zymography. A comparison of the N-terminal amino acid sequence between cColH and rColH revealed that rColH has 10 extra N-terminal amino acid residues. However, the peptide mapping of rColH with V8 protease was virtually identical to that of cColH. Furthermore, the molecular mass of rColH was estimated to be 112,999 Da by mass spectrometry, coinciding with the value of 112,977 Da, which was predicted from the nucleotide sequence of the colH gene. Therefore, the recombinant B. subtilis culture is capable of serving as a useful source for enzyme purification.

Identification of the gene encoding a mechanosensitive channel MscL homologue in Clostridium perfringens.

The mscL gene, which encodes the protein forming a large-conductance mechanosensitive channel (MscL) in Escherichia coli, has previously been cloned and sequenced by Sukharev et al. [Nature 368 (1994) 265-268]. We found a gene homologous to mscL in Clostridium perfringens which is located just downstream from the collagenase-encoding gene in the opposite direction.

A reduction in allergen-induced Fc epsilon R2/CD23 expression on peripheral B cells correlates with successful hyposensitization in grass pollinosis.

BACKGROUND: The cellular basis for the mechanism of specific hyposensitization is still unclear. OBJECTIVE: We prospectively studied the effect of immunotherapy on allergen-induced proliferation and Fc epsilon R2/CD23 expression of lymphocytes. METHODS: Mononuclear cells prepared from the peripheral blood of 22 patients with grass pollen (GP) allergy before, during, and after a preseasonal immunotherapy period with GP were stimulated with GP or control antigens. Tritiated thymidine uptake and percentage of CD23+ B cells were determined daily during days 6 to 8 and compared with lymphocyte responsiveness of 11 only symptomatically treated atopic patients and 14 nonatopic individuals. RESULTS: GP-induced lymphocyte proliferative response of both hyposensitized and symptomatically treated GP-allergic patients decreased markedly before the pollen season and rose again after seasonal allergen exposure, whereas a long-lived decrease in GP-induced Fc epsilon R2/CD23+ B cells was only observed in GP-treated patients. Alterations in Fc epsilon R2/CD23 expression were closely related to changes in symptoms and medication requirement during the following pollen season. In contrast, immunotherapy had no effect on Fc epsilon R2/CD23 expression of B cells without stimulation or on B cells cultured in the presence of control antigens. CONCLUSION: Because Fc epsilon R2/CD23 expression on B cells is antagonistically regulated by the cytokines interleukin-4 and interferon-gamma, the decrease of allergen-induced Fc epsilon R2/CD23+ B cells indicates an altered cytokine secretion pattern of the allergen-specific T lymphocytes with a predominance of interferon-gamma.

Detection of both isotypes of complement C4, C4A and C4B, in normal human glomeruli.

Monoclonal antibodies reactive against the complement C4A and C4B isotypic components were used in an immunoperoxidase technique for the histological study of normal human renal tissue. Prominent staining with both antibodies was seen in the mesangial areas of all normal kidney sections investigated. Occasional staining of arteriolar walls of the same tissues, however, was also observed. In contrast, no mesangial staining was seen using monoclonal antibodies reactive against other 'early' complement components, such as C1q and C3. Specificity of the glomerular staining with the anti-C4 reagents was demonstrated in two patients possessing only the C4A serum component but lacking genetically the C4B locus products. As would be predicted, glomerular staining with the anti-C4A reagent, but not anti-C4B, was clearly demonstrable. It is concluded that both isotypes of complement C4 are present in normal human glomeruli and thus might be operative for normal mesangial function.