Fluorescence anisotropy studies on the Ku-DNA interaction: anion and cation effects

DNA non-homologous end joining starts with the binding of Ku heterodimers to double strand breaks. In this work, we characterized the thermodynamics of the Ku-DNA interaction by fluorescence anisotropy of the probe-labeled DNA. We determined that the microscopic dissociation constant (kd) for the binding of Ku to a DNA binding site of the proper length (>20 bp) ranges from 22 to 29 nm at 300 mm NaCl. The binding isotherms for DNA duplexes with two or three heterodimers were analyzed with two independent models considering the presence and absence of overlapping binding sites. This analysis demonstrated that there is no or very weak nearest-neighbor cooperativity among the Ku molecules. These models can most likely be applied to study the interaction of Ku with duplexes of any length. Furthermore, our salt dependence studies indicated that electrostatic interactions play a major role in the binding of Ku to DNA and that the kd decreases approximately 60-fold as the salt concentration is lowered from 300 to 200 mm. The slope (Gammasalt) of the plot of log kd versus log[NaCl] is 12.4 +/- 0.1. This value is among the highest reported in the literature for a protein-DNA interaction and suggests that approximately 12 ions are released upon formation of the Ku-DNA complex. In addition, comparison of the slope values measured upon varying the type of cation and anion indicated that approximately nine cations and three anions are released from DNA and Ku, respectively, when the complex is formed.     

Comparison of different primer sets for use in automated ribosomal intergenic spacer analysis of complex bacterial communities

ITSF and ITSReub, constituting a new primer set designed for the amplification of the 16S-23S rRNA intergenic transcribed spacers, have been compared with primer sets consisting of 1406F and 23Sr (M. M. Fisher and E. W. Triplett, Appl. Environ. Microbiol. 65:4630-4636, 1999) and S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 (L. Ranjard et al., Appl. Environ. Microbiol. 67:4479-4487, 2001), previously proposed for automated ribosomal intergenic spacer analysis (ARISA) of complex bacterial communities. An agricultural soil and a polluted soil, maize silage, goat milk, a small marble sample from the facade of the Certosa of Pavia (Pavia, Italy), and brine from a deep hypersaline anoxic basin in the Mediterranean Sea were analyzed with the three primer sets. The number of peaks in the ARISA profiles, the range of peak size (width of the profile), and the reproducibility of results were used as indices to evaluate the efficiency of the three primer sets. The overall data showed that ITSF and ITSReub generated the most informative (in term of peak number) and reproducible profiles and yielded a wider range of spacer sizes (134 to 1,387) than the other primer sets, which were limited in detecting long fragments. The minimum amount of DNA template and sensitivity in detection of minor DNA populations were evaluated with artificial mixtures of defined bacterial species. ITSF and ITSReub amplified all the bacteria at DNA template concentrations from 280 to 0.14 ng microl(-1), while the other primer sets failed to detect the spacers of one or more bacterial strains. Although the primer set consisting of ITSF and ITSReub and that of S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 showed similar sensitivities for the DNA of Allorhizobium undicula mixed with the DNA of other species, the S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 primer set failed to detect the DNA of Pseudomonas stutzeri.     

Monthly dynamics of ticks (Acari: Ixodida) infesting N'Dama cattle in the Republic of Guinea

The occurrence of ixodid ticks on N'Dama cattle was studied in the Republic of Guinea between June 1994 and May 1995. Monthly tick collections were performed on 80 animals from 14 villages located in Dabola, Kouroussa and Dinguiraye prefectures. A total of 19,804 ticks was collected and classified using standard taxonomic keys. The following tick species were identified: Amblyomma variegatum, Hyalomma marginatum rufipes, Hyalomma trunctum, Hyalomma nitidum, Rhipicephalus lunulatus, Rhipicephalus muhsamae, Rhipicephalus senegalensis, Rhipicephalus sulcatus, Rhipicephalus turanicus, Boophilus annulatus, Boophilus geigyi. Boophilus spp. were the most numerous adult ticks (57.1%), Am. variegatum adults constituted 27.4%, while 12.4% were Rhipicephalus spp. and 2.5% Hyalomma spp. Rhipicephalus turanicus and Hyalomma nitidum were recorded for the first time in the country. Am. variegatum and Boophilus spp. were present throughout the year, whereas Am. variegatum adults showed a peak during the rainy season between April and September. Immature stages collected belonged exclusively to the genera Amblyomma and Boophilus. Am. variegatum larvae and nymphs showed a peak during the dry season (October-March); no significant variation between seasons was observed for Boophilus immatures. A significantly higher infestation of cattle by Rhipicephalus spp. was found in Dabola and Kouroussa prefectures, located in the southern part of the study area, with similar climatic, vegetation and rainfall characteristics. Possible options for the control of ticks in the study area are briefly discussed.     

Heterogenic response of the liver parenchyma to ethanol studied in the bivascularly perfused rat liver

Zonation of ethanol oxidation and metabolic effects along the hepatic acini were investigated in the bivascularly perfused liver of fed rats. Ethanol was infused into the hepatic artery in antegrade and retrograde perfusion. Inhibition of glycolysis by ethanol, expressed as micromol min(-1) (ml accessible cell space)(-1), was more pronounced in the retrograde mode; the retrograde/antegrade ratio was equal to 1.63 for an ethanol infusion rate of 37.5 micromol min(-1) g(-1). Stimulation of oxygen uptake by ethanol was more pronounced in the retrograde mode; the retrograde/antegrade ratio was equal to 1.77. Diminution of the citrate cycle caused by ethanol was more pronounced in the retrograde mode; the retrograde/antegrade ratio was equal to 1.46. Transformation of arterially infused ethanol into acetate was more pronounced in retrograde perfusion; the retrograde/antegrade ratio was equal to 1.63. The increments in glucose release (glycogenolysis) caused by ethanol in the antegrade and retrograde modes were similar. It was assumed that the changes caused by arterially infused ethanol in retrograde and antegrade perfusion closely reflect a significant part of the periportal parenchyma and an average over the whole liver parenchyma, respectively. Under such assumptions it can be concluded that, in the perfused liver from fed rats, four related parameters predominate in the periportal region: ethanol oxidation, glycolysis inhibition, oxygen uptake stimulation and citrate cycle inhibition. One of the main causes for this predominance could be the malate/aspartate shuttle, which operates more rapidly in the periportal area and is essential for NADH oxidation.     

F0F1 ATP synthase activity is differently modulated by coronary reactive hyperemia before and after ischemic preconditioning in the goat

The amplitude of coronary reactive hyperemia (CRH), elicited by 15 s of ischemia, is reduced in hearts subjected to 5 min of ischemic preconditioning (IP). F0F1 ATP synthase activity and ATP concentration are also altered by IP. We hypothesized that F0F1 ATP synthase is differently modulated by the inhibitor protein IF(1) during CRH elicited before (CRHnp) and after (CRHprec) IP. Hemodynamic parameters were recorded in 10 anesthetized goats. Myocardial biopsies were obtained before IP (Cnp), during CRHnp, 4 and 6 min after the onset of CRHnp, after IP (Cprec), during CRHprec, and 4 min after CRHprec. F0F1 ATP synthase activity, ATP concentration, and ATP-to-ADP ratio (ATP/ADP) were determined. Compared with CRHnp, IP blunted CRHprec. F0F1 ATP synthase activity transiently increased during CRHnp, decreased 4 min after CRHnp, and returned to control 2 min later; it was lower after IP (Cprec) and did not change during and after CRHprec. All these changes in activity were modulated by IF1. During CRHnp, ATP concentration and ATP/ADP were reduced compared with Cnp and began to rise 6 min thereafter. During Cprec, both parameters were transiently reduced but increased during and after CRHprec. Hence, during CRHnp, F0F1 ATP synthase activity transiently increases and then decreases significantly. The short-lasting inhibition of the enzyme may explain why a few seconds of occlusion do not induce IP. After IP, F0F1 ATP synthase activity is blunted, and it is not affected by a subsequent 15 s of occlusion, which induces a blunted CRHprec. These results suggest that postischemic long-lasting inhibition of F0F1 ATP synthase activity may be a feature of the preconditioned heart. The increase in ATP concentration after preconditioning is in agreement with previous reports of reduced ATP hydrolysis by cytoplasmic ATPases.     

Effect of stent coating alone on in vitro vascular smooth muscle cell proliferation and apoptosis

Synthetic polymers, like methacrylate (MA) compounds, have been clinically introduced as inert coatings to locally deliver drugs that inhibit restenosis after stent. The aim of the present study was to evaluate the effects of MA coating alone on vascular smooth muscle cell (VSMC) growth in vitro. Stainless steel stents were coated with MA at the following doses: 0.3, 1.5, and 3 ml. Uncoated/bare metal stents were used as controls. VSMCs were cultured in dishes, and a MA-coated stent or an uncoated bare metal stent was gently added to each well. VSMC proliferation was assessed by bromodeoxyuridine (BrdU) incorporation. Apoptosis was analyzed by three distinct approaches: 1) annexin V/propidium iodide fluorescence detection; 2) DNA laddering; and 3) caspase-3 activation and PARP cleavage. MA-coated stents induced a significant decrease of BrdU incorporation compared with uncoated stents at both the low and high concentrations. In VSMCs incubated with MA-coated stents, annexin V/propidium iodide fluorescence detection showed a significant increase in apoptotic cells, which was corroborated by the typical DNA laddering. Apoptosis of VSMCs after incubation with MA-coated stents was characterized by caspase-3 activation and PARP cleavage. The MA-coated stent induced VSMC growth arrest by inducing apoptosis in a dose-dependent manner. Thus MA is not an inert platform for eluting drugs because it is biologically active per se. This effect should be taken in account when evaluating an association of this coating with antiproliferative agents for in-stent restenosis prevention.     

Isolation of Elaeagnus-compatible Frankia from soils collected in Tunisia

The occurrence and diversity of Frankia nodulating Elaeagnus angustifolia in Tunisia were evaluated in 30 soils from different regions by a Frankia-capturing assay. Despite the absence of actinorhizal plants in 24 of the 30 soils, nodules were captured from all the samples. Eight pure strains were isolated from single colonies grown in agar medium. On the basis of 16S rRNA and GlnII sequences, seven strains were clustered with Frankia, colonizing Elaeagnaceae and Rhamnaceae in two different phylogenetic groups while one strain described a new lineage in the Frankia assemblage, indicating that Frankia strains genetically diverse from previously known Elaeagnus-infective strains are present in tunisian soils. Genomic fingerprinting determined by rep-PCR, and tDNA-PCR-SSCP, confirmed the wide genetic diversity of the strains.     

Efficient recruitment of lymphocytes in inflamed brain venules requires expression of cutaneous lymphocyte antigen and fucosyltransferase-VII

Lymphocyte migration into the brain represents a critical event in the pathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). However, the mechanisms controlling the recruitment of lymphocytes to the CNS via inflamed brain venules are poorly understood, and therapeutic approaches to inhibit this process are consequently few. In this study, we demonstrate for the first time that human and murine Th1 lymphocytes preferentially adhere to murine inflamed brain venules in an experimental model that mimics early inflammation during EAE. A virtually complete inhibition of rolling and arrest of Th1 cells in inflamed brain venules was observed with a blocking anti-P-selectin glycoprotein ligand 1 Ab and anti-E- and P-selectin Abs. Th1 lymphocytes produced from fucosyltransferase (FucT)-IV(-/-) mice efficiently tethered and rolled, whereas in contrast, primary adhesion of Th1 lymphocytes obtained from FucT-VII(-/-) or Fuc-VII(-/-)FucT-IV(-/-) mice was drastically reduced, indicating that FucT-VII is critical for the recruitment of Th1 cells in inflamed brain microcirculation. Importantly, we show that Abs directed against cutaneous lymphocyte Ag (CLA), a FucT-VII-dependent carbohydrate modification of P-selectin glycoprotein ligand 1, blocked rolling of Th1 cells. By exploiting a system that allowed us to obtain Th1 and Th2 cells with skin- vs gut-homing (CLA(+) vs integrin beta(7)(+)) phenotypes, we observed that induced expression of CLA on Th cells determined a striking increase of rolling efficiency in inflamed brain venules. These observations allow us to conclude that efficient recruitment of activated lymphocytes to the brain in the contexts mimicking EAE is controlled by FucT-VII and its cognate cell surface Ag CLA.