Induction of AT-specific DNA-interstrand crosslinks by bizelesin in genomic and simian virus 40 DNA.
Bizelesin is a bifunctional AT-specific DNA alkylating drug. Our study characterized the ability of bizelesin to induce interstrand crosslinks, a potential lethal lesion. In genomic DNA of BSC-1 cells, bizelesin formed from approx. 0.3 to 6.03+/-0.85 interstrand crosslinks per 106 base pairs, at 5-100 nM drug concentration, respectively, comparable to the number of total adducts previously determined in the same system (J.M. Woynarowski, M.M. McHugh, L.S. Gawron, T.A. Beerman, Biochemistry 34 (1995) 13042-13050). Bizelesin did not induce DNA-protein crosslinks or strand breaks. A model defined target, intracellular simian virus 40 (SV40) DNA, was employed to map at the nucleotide level sites of bizelesin adducts, including potential interstrand crosslinks. Preferential adduct formation was observed at AT tracts which are abundant in the SV40 matrix associated region and the origin of replication. Many sites, including each occurrence of 5'-T(A/T)4A-3', co-mapped on both DNA strands suggesting interstrand crosslinks, although monoadducts were also formed. Bizelesin adducts in naked SV40 DNA were found at similar sites. The localization of bizelesin-induced crosslinks in AT-rich tracts of replication-related regions is consistent with the potent anti-replicative properties of bizelesin. Given the apparent lack of other types of lesions in genomic DNA, interstrand crosslinks localized in AT-rich tracts, and to some extent perhaps also monoadducts, are likely to be lethal effects of bizelesin. (+info)
Separation and properties of two acetylacetoin reductases from Bacillus cereus YUF-4.
The separation and purification of two kinds of acetylacetoin reductases (AACRs) from Bacillus cereus YUF-4 were examined. NADPH-linked AACR (AACR I) and NADH-linked AACR (AACR II) were separated from each other by ammonium sulfate fractionation, DEAE-cellulose chromatography, and hydroxyapatite chromatography. The former was purified 3.4-fold with a yield of 10.0%, and the latter was purified 29-fold with a yield of 15.6%. The two enzymes differ from each other in some enzymic properties such as substrate specificity. (+info)
Two types of HTLV-1 particles are released from MT-2 cells.
The MT-2 cell line transformed by human T-cell leukemia virus type 1 (HTLV-1) contains one complete provirus and seven defective proviruses. Four defective genomes have an identical structure (LTR-MA-deltaCA-pX-LTR) with an open reading frame that spans from MA to pX, giving rise to a 3.4-kb (24S) RNA transcript encoding a chimeric Gag-pX protein, p28. MT-2 cells release two distinct types of virions. The major "classic" type of particle has a buoyant density of 1.155-1.16 g/cm3 and contains the standard HTLV-I structural proteins and reverse transcriptase (RT). In addition, about 5% of particles are "light," approximately 1.12 g/cm3, and contain p28, RT activity, and the 3.4-kb RNA transcript. RT-PCR and in vitro translation indicate that some of the classic HTLV-1 particles package 3.4-kb RNA as well as full-length 8.5-kb RNA. In addition to matrix features, the p28 protein has a motif resembling a zinc finger at the C-terminal, pX0 region, which may play a role in the assembly of the defective light virions. (+info)
Localization of a candidate surfactant convertase to type II cells, macrophages, and surfactant subfractions.
Pulmonary surfactant exists in the alveolus in several distinct subtypes that differ in their morphology, composition, and surface activity. Experiments by others have implicated a serine hydrolase in the production of the inactive small vesicular subtype of surfactant (N. J. Gross and R. M. Schultz. Biochim. Biophys. Acta 1044: 222-230, 1990). Our laboratory recently identified this enzyme in the rat as the serine carboxylesterase ES-2 [F. Barr, H. Clark, and S. Hawgood. Am. J. Physiol. 274 (Lung Cell. Mol. Physiol. 18): L404-L410, 1998]. In the present study, we determined the cellular sites of expression of ES-2 in rat lung using a digoxygenin-labeled ES-2 riboprobe. ES-2 mRNA was localized to type II cells and alveolar macrophages but not to Clara cells. Using a specific ES-2 antibody, we determined the protein distribution of ES-2 in the lung by immunohistochemistry, and it was found to be consistent with the sites of mRNA expression. Most of the ES-2 in rat bronchoalveolar lavage is in the surfactant-depleted supernatant, but ES-2 was also consistently localized to the small vesicular surfactant subfraction presumed to form as a consequence of conversion activity. These results are consistent with a role for endogenous lung ES-2 in surfactant metabolism. (+info)
Inhibition of protein denaturation by fatty acids, bile salts and other natural substances: a new hypothesis for the mechanism of action of fish oil in rheumatic diseases.
Natural hydrophobic substances like bile salts (cholate, deoxycholate, chenodeoxycholate, lithocholate and their conjugates with glycine and taurine), fatty acids (caprylic, capric, lauric, myristic, palmitic, stearic, oleic, linoleic, arachidonic, eicosapentaenoic and docosahexaenoic acid) were much more active (EC50 approximately 10(-4)-10(-5) M) than selected amino acids (EC50 > 10(-2) M) and inorganic salts (EC50 approximately 10(-1) M) in inhibiting heat-induced denaturation of human serum albumin in vitro. Fish oil, rich in n-3-polyunsaturated acids such as eicosapentaenoic acid and docosahexaenoic acid, administered p.o. (1 ml/kg) in the rat, protected ex vivo (after 2 hr) serum against heat-induced denaturation more than bendazac, a known antidenaturant drug. Thus, we speculated that the antidenaturant activity of fish oil may be partly (in addition to the known effect on endogenous eicosanoid composition) responsible for its beneficial effects in rheumatoid arthritis and other rheumatic conditions. In this connection, it is of note that the in vitro antidenaturant activity of fish oil fatty acids was higher than that of known antidenaturant drugs such as bendazac and bindarit and nonsteroidal anti-inflammatory drugs like phenylbutazone and indomethacin which could exert beneficial effects in chronic inflammatory conditions by stabilizing endogenous proteins. (+info)
European interlaboratory comparison of breath 13CO2 analysis.
The BIOMED I programme Stable Isotopes in Gastroenterology and Nutrition (SIGN) has focused upon evaluation and standardisation of stable isotope breath tests using 13C labelled substrates. The programme dealt with comparison of 13C substrates, test meals, test conditions, analysis techniques, and calculation procedures. Analytical techniques applied for 13CO2 analysis were evaluated by taking an inventory of instrumentation, calibration protocols, and analysis procedures. Two ring tests were initiated measuring 13C abundances of carbonate materials. Evaluating the data it was found that seven different models of isotope ratio mass spectrometers (IRMS) were used by the participants applying both the dual inlet system and the continuous flow configuration. Eight different brands of certified 13C reference materials were used with a 13C abundance varying from delta 13CPDB -37.2 to +2.0/1000. CO2 was liberated from certified material by three techniques and different working standards were used varying from -47.4 to +0.4/1000 in their delta 13CPDB value. The standard deviations (SDs) found for all measurements by all participants were 0.25/1000 and 0.50/1000 for two carbonates used in the ring tests. The individual variation for the single participants varied from 0.02 /1000 (dual inlet system) to 0.14/1000 (continuous flow system). The measurement of the difference between two carbonates showed a SD of 0.33/1000 calculated for all participants. Internal precision of IRMS as indicated by the specifications of the different instrument suppliers is < 0.3/1000 for continuous flow systems. In this respect it can be concluded that all participants are working well within the instrument specifications even including sample preparation. Increased overall interlaboratory variation is therefore likely to be due to non-instrumental conditions. It is possible that consistent differences in sample handling leading to isotope fractionation are the causes for interlaboratory variation. Breath analysis does not require sample preparation. As such, interlaboratory variation will be less than observed for the carbonate samples and within the range indicated as internal precision for continuous flow instruments. From this it is concluded that pure analytical interlaboratory variation is acceptable despite the many differences in instrumentation and analytical protocols. Coordinated metabolic studies appear possible, in which different European laboratories perform 13CO2 analysis. Evaluation of compatibility of the analytical systems remains advisable, however. (+info)
Lipopolysaccharides (LPS) of oral black-pigmented bacteria induce tumor necrosis factor production by LPS-refractory C3H/HeJ macrophages in a way different from that of Salmonella LPS.
Some lipopolysaccharide (LPS) preparations from S- or R-form members of the family Enterobacteriaceae and oral black-pigmented bacteria (Porphyromonas gingivalis and Prevotella intermedia) are known to activate LPS-refractory C3H/HeJ macrophages. When contaminating proteins are removed from R-form LPS of Enterobacteriaceae by repurification, however, this ability is lost. In the present study, we investigated the capacity of LPS from P. gingivalis, P. intermedia, Salmonella minnesota, and Salmonella abortusequi to induce production of tumor necrosis factor (TNF) in gamma interferon-primed C3H/HeJ macrophages before and after repurification. P. abortusequi S-LPS was fractionated by centrifugal partition chromatography into two LPS forms: SL-LPS, having homologous long O-polysaccharide chains, and SS-LPS having short oligosaccharide chains. Prior to repurification, all LPS forms except SL-LPS induced TNF production in both C3H/HeJ and C3H/HeN macrophages. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that repurification removed contaminating protein from the preparations, and repurified SS-LPS and S. minnesota Ra-LPS no longer stimulated TNF production in C3H/HeJ macrophages, although C3H/HeN macrophages remained responsive. In contrast, repurified oral bacterial LPS retained the capacity to induce TNF production in C3H/HeJ macrophages. Oral bacterial LPS preparations also were not antagonized by excess inactive, repurified SL-LPS; Ra-LPS; Rhodobacter sphaeroides lipid A, a competitive LPS antagonist, or paclitaxel, an LPS agonist, and they were comparatively resistant to polymyxin B treatment. Nevertheless, oral bacterial LPS was less toxic to D-galactosamine-treated C3H/HeN mice than was LPS from Salmonella. These findings indicate that the active molecule(s) and mode of action of LPS from P. gingivalis and P. intermedia are quite different from those of LPS from Salmonella. (+info)
Characterization of a novel trypanosome lytic factor from human serum.
Natural resistance of humans to the cattle pathogen Trypanosoma brucei brucei has been attributed to the presence in human serum of nonimmune factors that lyse the parasite. Normal human serum contains two trypanosome lytic factors (TLFs). TLF1 is a 500-kDa lipoprotein, which is reported to contain apolipoprotein A-I (apoA-I), haptoglobin-related protein (Hpr), hemoglobin, paraoxonase, and apoA-II, whereas TLF2 is a larger, poorly characterized particle. We report here a new immunoaffinity-based purification procedure for TLF2 and TLF1, as well as further characterization of the components of each purified TLF. Immunoaffinity-purified TLF1 has a specific activity 10-fold higher than that of TLF1 purified by previously described methods. Moreover, we find that TLF1 is a lipoprotein particle that contains mainly apoA-I and Hpr, trace amounts of paraoxonase, apoA-II, and haptoglobin, but no detectable hemoglobin. Characterization of TLF2 reveals that it is a 1,000-kDa protein complex containing mainly immunoglobulin M, apoA-I, and Hpr but less than 1% detectable lipid. (+info)