Efficiency of DNA-histone crosslinking induced by saturated and unsaturated aldehydes in vitro.

Using a filter-binding assay based on precipitation of pUC13 plasmid DNA bound to calf-thymus histones, we have determined the efficiency of formation of DNA-protein crosslink formation induced by several aldehyde compounds in vitro. Formaldehyde, glutaraldehyde and acrolein were the most potent, causing 1 crosslink per 2.7 kbp of DNA at 1.5, 8 and 150 microM, respectively. All other compounds tested gave 1 crosslink per plasmid molecule in the mM concentration range as follows: acetaldehyde, 115 mM; propionaldehyde, 295 mM; butyraldehyde, 360 mM; crotonaldehyde, 8.5 mM; trans-2-pentenal, 6.3 mM. Significant decreases in the efficiency of DPXL formation were observed with monofunctional aldehydes of higher carbon chain length. For example, the concentration of formaldehyde needed to give 1 crosslink per molecule was almost 10(5) times less than that of acetaldehyde. Acetaldehyde differs from formaldehyde only by one saturated carbon. The presence of an unsaturated bond between the 2-3 carbons improved the potential for crosslink formation. For example, acrolein was over 500-fold more potent than propionaldehyde. Glutaraldehyde was almost as potent as formaldehyde, indicating that the bifunctional nature of this 5-carbon saturated aldehyde may be crucial to its high efficiency of DNA-protein crosslinking.     

Genetic diversity among Rhizobium strains from Cicer arietinum L.

Following bacteriological cloning and determination of their symbiotic performance, 15 representative, diverse strains of chickpea rhizobia were genetically analysed for restriction fragment length polymorphisms. Analyses of genomic DNAs showed several different groups. Almost half (7) of the strains examined were very similar and clearly represented a single species. There was a related group of four strains which could be a subspecies. There was also one distinct group of four strains which were apparently unrelated to the reference strain 3377. This latter group may constitute a separate species. Phenotypic differences should be investigated further.     

Biochemical and symbiotic properties of histidine-requiring mutants of Rhizobium leguminosarum biovar trifolii

A perturbation of the histidine biosynthetic pathway in legume microsymbionts can abolish their symbiotic competence. Twenty-one histidine-requiring (His⁻) mutants were isolated from berseem clover-nodulating, symbiotically-competent (Nod⁺, Fix⁺) Rhizobium leguminosarum bv. ' trifolii ' strain RTH 48 Sm^r by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis followed by enrichment. These mutants were analysed for their biochemical defect and the corresponding effect, if any, on their symbiotic abilities. Cross-feeding, supplementation and enzymatic studies identified three types of mutants. Group 1 mutants, His-2 and His-12, grew with histidine supplementation but not with the addition of either L -histidinol or L -histidinol phosphate to the medium ; they lacked histidinol dehydrogenase (EC 1.1.1.23) activity and consequently formed only ineffective, or 'non-fixing' nodules. Group 2 mutant, His-17, grew when supplemented with either L -histidinol or L -histidine, had low histidinol phosphate phosphatase (EC 3.1.3.15) activity (37% of wild-type), and consequently failed to nodulate berseem clover. Group 3, the remaining 18 mutants, grew when supplemented with L -histidinol phosphate, L -histidinol or histidine, and did not nodulate. Typically, reversion rates were between 10⁻⁷ and 10⁻⁸. Defects in early steps of the pathway abolished nodulating ability, whereas lesions in the last step did not. The last step, however, was required for symbiotic nitrogen fixation. It is hypothesized that histidine may be supplied by the host in sufficient quantity for nodulation by histidinol dehydrogenase mutants to occur, whereas the amount provided in the nodule may be insufficient to support bacteroid development and nitrogen fixation.     

Inhibition of matrix metalloproteinase-9 secretion by dimethyl sulfoxide and cyclic adenosine monophosphate in human monocytes

BACKGROUND Matrix metalloproteinases(MMPs),including MMP-9,are an integral part of the immune response and are upregulated in response to a variety of stimuli.New details continue to emerge concerning the mechanistic and regulatory pathways that mediate MMP-9 secretion.There is significant evidence for regulation of inflammation by dimethyl sulfoxide(DMSO)and 3',5'-cyclic adenosine monophosphate(cAMP),thus investigation of how these two molecules may regulate both MMP-9 and tumor necrosis factorα(TNFα)secretion by human monocytes was of high interest.The hypothesis tested in this study was that DMSO and cAMP regulate MMP-9 and TNFαsecretion by distinct mechanisms.AIM To investigate the regulation of lipopolysaccharide(LPS)-stimulated MMP-9 and tumor necrosis factorαsecretion in THP-1 human monocytes by dimethyl sulfoxide and cAMP.METHODS The paper describes a basic research study using THP-1 human monocyte cells.All experiments were conducted at the University of Missouri-St.Louis in the Department of Chemistry and Biochemistry.Human monocyte cells were grown,cultured,and prepared for experiments in the University of Missouri-St.Louis Cell Culture Facility as per accepted guidelines.Cells were treated with LPS for selected exposure times and the conditioned medium was collected for analysis of MMP-9 and TNFαproduction.Inhibitors including DMSO,cAMP regulators,and anti-TNFαantibody were added to the cells prior to LPS treatment.MMP-9 secretion was analyzed by gel electrophoresis/western blot and quantitated by ImageJ software.TNFαsecretion was analyzed by enzyme-linked immuno sorbent assay.All data is presented as the average and standard error for at least 3 trials.Statistical analysis was done using a two-tailed paired Student t-test.P values less than 0.05 were considered significant and designated as such in the Figures.LPS and cAMP regulators were from Sigma-Aldrich,MMP-9 standard and antibody and TNFαantibodies were from R&D Systems,and amyloid-βpeptide was from rPeptide.RESULTS In our investigation of MMP-9 secretion from THP-1 human monocytes,we made the following findings.Inclusion of DMSO in the cell treatment inhibited LPSinduced MMP-9,but not TNFα,secretion.Inclusion of DMSO in the cell treatment at different concentrations inhibited LPS-induced MMP-9 secretion in a dosedependent fashion.A cell-permeable cAMP analog,dibutyryl cAMP,inhibited both LPS-induced MMP-9 and TNFαsecretion.Pretreatment of the cells with the adenylyl cyclase activator forskolin inhibited LPS-induced MMP-9 and TNFαsecretion.Pretreatment of the cells with the general cAMP phosphodiesterase inhibitor IBMX reduced LPS-induced MMP-9 and TNFαin a dose-dependent fashion.Pre-treatment of monocytes with an anti-TNFαantibody blocked LPSinduced MMP-9 and TNFαsecretion.Amyloid-βpeptide induced MMP-9 secretion,which occurred much later than TNFαsecretion.The latter two findings strongly suggested an upstream role for TNFαin mediating LPS-stimulate MMP-9 secretion.CONCLUSION The cumulative data indicated that MMP-9 secretion was a distinct process from TNFαsecretion and occurred downstream.First,DMSO inhibited MMP-9,but not TNFα,suggesting that the MMP-9 secretion process was selectively altered.Second,cAMP inhibited both MMP-9 and TNFαwith a similar potency,but at different monocyte cell exposure time points.The pattern of cAMP inhibition for these two molecules suggested that MMP-9 secretion lies downstream of TNFαand that TNFαmay a key component of the pathway leading to MMP-9 secretion.This temporal relationship fit a model whereby early TNFαsecretion directly led to later MMP-9 secretion.Lastly,antibody-blocking of TNFαdiminished MMP-9 secretion,suggesting a direct link between TNFαsecretion and MMP-9 secretion.     

2-Aminobenzamide, an inhibitor of ADP-ribosylation, antagonizes induced DNA hypomethylation during differentiation of murine Friend erythroleukemia cells by N''-methylnicotinamide

The ADP-ribose synthesis inhibitor 2-aminobenzamide was found to reduce the induction of haemoglobin synthesis in murine Friend erythroleukemia cells, cultured continuously for 96 h with 5 mM N'-methylnicotinamide, with over 50% inhibition at equimolar doses. 2-Aminobenzamide was optimally effective as an inhibitor of differentiation if present only during the time period 24-48 h after initial N'-methylnicotinamide exposure. A transient, genome-wide DNA hypomethylation accompanies induction by N'-methylnicotinamide. Although 2-aminobenzamide does not seem to affect normal DNA methylation in cultured cells, the observed DNA hypomethylation in cells cultured for 24 h with 5 mM N'-methylnicotinamide was antagonized over 50% by equimolar 2-aminobenzamide. We suggest that ADP-ribosylation has a positive modulatory role in erythroid differentiation, and possibly in the process(es) leading to DNA hypomethylation following inducer exposure.