Incorporation of vitronectin into fibrin clots. Evidence for a binding interaction between vitronectin and gamma A/gamma' fibrinogen.

Vitronectin is an abundant plasma protein that regulates coagulation, fibrinolysis, complement activation, and cell adhesion. Recently, we demonstrated that plasma vitronectin inhibits fibrinolysis by mediating the interaction of type 1 plasminogen activator inhibitor with fibrin (Podor, T. J., Peterson, C. B., Lawrence, D. A., Stefansson, S., Shaughnessy, S. G., Foulon, D. M., Butcher, M., and Weitz, J. I. (2000) J. Biol. Chem. 275, 19788-19794). The current studies were undertaken to further examine the interactions between vitronectin and fibrin(ogen). Comparison of vitronectin levels in plasma with those in serum indicates that approximately 20% of plasma vitronectin is incorporated into the clot. When the time course of biotinylated-vitronectin incorporation into clots formed from (125)I-fibrinogen is monitored, vitronectin incorporation into the clot parallels that of fibrinogen in the absence or presence of activated factor XIII. Vitronectin binds specifically to fibrin matrices with an estimated K(d) of approximately 0.6 microm. Additional vitronectin subunits are assembled on fibrin-bound vitronectin multimers through self-association. Confocal microscopy of fibrin clots reveals the globular vitronectin aggregates anchored at intervals along the fibrin fibrils. This periodicity raised the possibility that vitronectin interacts with the gamma A/gamma' variant of fibrin(ogen) that represents about 10% of total fibrinogen. In support of this concept, the vitronectin which contaminates fibrinogen preparations co-purifies with the gamma A/gamma' fibrinogen fraction, and clots formed from gamma A/gamma' fibrinogen preferentially bind vitronectin. These studies reveal that vitronectin associates with fibrin during coagulation, and may thereby modulate hemostasis and inflammation.     

Development of H1e histone linker-specific antibodies by means of synthetic peptides.

A large body of data suggests that the linker histones family (H1) affects gene expression. Investigation of the linker histones role is then of a major interest in cell cycle studies with implications in gene therapy. Indeed, it has been shown that in most tissues a switch of histone subtypes occurs when the cells cease to divide. To investigate linker histone role in gene or transgene expression, an antibody against subtypes of H1 would be useful for immunoprecipitation experiments and further assays measuring H1subtypes-DNA interactions in living cells. In order to produce an antibody against the H1e subtype of linker histones, two synthetic peptides derived from two regions of the H1e mouse histone protein were examined for their potential, [as keyhole limpet hemocyanin (KLH) conjugates] to elicit polyclonal anti-H1e antibodies in New Zealand white rabbits. Selection of the peptide sequences was based on amino acid differences within the different classes of histones and between mice and rabbit histones as well. The evaluation of their potential immunogenic properties was based on examination of peptide hydropathy using predicting algorithms. Immunoglobulins (IgG) obtained from immunized and nonimmunized rabbits were tested using enzyme-linked immunosorbent assay (ELISA) procedures, Western immunoblot, and immunofluorescence experiments. Results showed that the selected synthetic peptides gave rise to a high-titer polyclonal antibody able to recognize the H1e histone under various conditions. This polyclonal antibody did not cross-react with other histones. To our knowledge, this is the first antibody produced against the mouse H1e linker histone.     

Ribosomal RNA secondary structure: compensatory mutations and implications for phylogenetic analysis

Using sequence data from the 28S ribosomal RNA (rRNA) genes of selected vertebrates, we investigated the effects that constraints imposed by secondary structure have on the phylogenetic analysis of rRNA sequence data. Our analysis indicates that characters from both base-pairing regions (stems) and non-base-pairing regions (loops) contain phylogenetic information, as judged by the level of support of the phylogenetic results compared with a well-established tree based on both morphological and molecular data. The best results (the greatest level of support of well-accepted nodes) were obtained when the complete data set was used. However, some previously supported nodes were resolved using either the stem or loop bases alone. Stem bases sustain a greater number of compensatory mutations than would be expected at random, but the number is < 40% of that expected under a hypothesis of perfect compensation to maintain secondary structure. Therefore, we suggest that in phylogenetic analyses, the weighting of stem characters be reduced by no more than 20%, relative to that of loop characters. In contrast to previous suggestions, we do not recommend weighting of stem positions by one-half, compared with that of loop positions, because this overcompensates for the constraints that selection imposes on the secondary structure of rRNA.      

Effect of an acute zinc depletion on rat lipoprotein distribution and peroxidation

The aim of this study was to determine the extent to which zinc depletion leads to lipoprotein modifications by measuring both lipoprotein-fraction distribution and peroxidation in zinc-depleted rats. The animals were divided into three groups and fed for 8 wk a zinc-adequate diet (100 ppm) ad libitum (AL), a zinc-deficient diet (0.2 ppm) ad libitum (ZD), or a zinc-adequate diet according to the pair feeding method (PF). Trace-element status, tissular lipids, and lipoprotein-fraction study were performed. The MDA production by the lipoprotein fraction was measured before and after induced peroxidation. Cholesterol and phospholipids were increased in ZD rats. An important increase of VLDL and IDL was observed and a significant enhanced production of MDA by the LDL was related to zinc deficiency. From this observation, we may conclude that LDL fractions of ZD rats are more susceptible to induced oxidative damage. These results suggest that in zinc deficiency, the lipoprotein fragility is an aggravating factor of peroxidation and the dyslipoproteinemia may lead to an atherogenic risk.     

New mutualistic fungal endophytes isolated from poplar roots display high metal tolerance

This study aimed to isolate, identify, and characterise metal-tolerant fungi colonising poplar roots at a metal-contaminated phytoremediation site. Poplar roots were colonised by arbuscular mycorrhizal, ectomycorrhizal, and endophytic fungi, and the species were determined by ITS molecular analyses. Eight different isolates were successfully isolated into pure culture. Three isolates belonging to the Helotiales (P02, P06) and the Serendipita vermifera species (P04) were highly tolerant to metals (Cd, Zn, Pb, and Cu) compared to the mycorrhizal Hebeloma isolates. The three isolates degraded complex carbohydrates, such as xylan and cellulose, indicating that they could partially degrade root cell walls and penetrate into cells. This hypothesis was confirmed by further in vitro re-synthesis experiments, which showed that the three isolates colonised root tissues of poplar plantlets whereas two of them formed microsclerotia-like structures. Taken together, these results suggest an endophytic lifestyle of these isolates. This is the first evidence of S. vermifera as a root endophyte of poplar. A new endophytic putative species belonging to the Helotiales and closely related to Leohumicola is also reported. Interestingly, and when compared to mock-inoculated plants, both P06 and P04 isolates increased the number of root tips of inoculated poplar plantlets in vitro. Moreover, the S. vermifera P04 isolate also increased the shoot biomass. The results are discussed in relation to the potential use of endophytic strains for tree-based phytoremediation of metal-contaminated sites.     

Abolition of ventilatory response to caffeine in chemodenervated lambs

In this study we have evaluated the role of the peripheral chemoreceptors in the ventilatory response to caffeine at a dose currently used in human infants for treatment of central apneas (10 mg/kg). Twelve lambs were studied; six had carotid body denervation (CBD) and six had a sham denervation (intact). The denervation was done the 2nd wk of life, and the study of the response to caffeine infusion was carried out at a mean age of 82 days. The awake and nonsedated animals received 10 mg/kg of caffeine, and caffeine blood levels were, respectively, 8.8 and 9.0 mg/l in the intact and in the CBD lambs. The intact lambs responded to caffeine by a significant immediate increase in minute ventilation (VE) of 46% from 274 to 400 ml X min-1 X kg-1 (P less than 0.001), 1 min after caffeine infusion. This response rapidly faded, but VE was still increased at 2 h, 314 ml X min-1 X kg-1. The increase in ventilation was brought about by a change in mean inspiratory flow (VT/TI), which increased from 9.9 to 14.0 ml X s-1 X kg-1 within 1 min (P less than 0.01); VT/TI was still increased at 11.2 ml X s-1 X kg-1 2 h later. In contrast, for the CBD lambs there was no response to caffeine infusion as measured by VE or VT/TI. We conclude that bolus caffeine infusion produces a rapid response in VE followed by a fall in VE that remained above base line until at least 2 h postinfusion, and the intact chemoreceptor function appears as an essential mediator for these increases in ventilation, since the peripheral chemodenervation has completely abolished the VE response to this particular dose of caffeine.     

New selective carbonic anhydrase IX inhibitors: Synthesis and pharmacological evaluation of diarylpyrazole-benzenesulfonamides

Carbonic anhydrase (CA) IX expression is increased upon hypoxia and has been proposed as a therapeutic target since it has been associated with poor prognosis, tumor progression and pH regulation. We report the synthesis and the pharmacological evaluation of a new class of human carbonic anhydrase (hCA) inhibitors, 4-(5-aryl-2-hydroxymethyl-pyrazol-1-yl)-benzenesulfonamides. A molecular modeling study was conducted in order to simulate the binding mode of this new family of enzyme inhibitors within the active site of hCA IX. Pharmacological studies revealed high hCA IX inhibitory potency in the parameters nanomolar range. This study showed that the position of sulfonamide group in meta of the 1-phenylpyrazole increase a selectivity hCA IX versus hCA II of our compounds. An in vitro antiproliferative screening has been performed on the breast cancer MDA-MB-231 cell using doxorubicin as cytotoxic agent and in presence of selected CA IX inhibitor. The results shown that the cytotoxic efficiency of doxorubicin in an hypoxic environment, expressed in IC₅₀ value, is restored at 20% level with 1 μM CA IX inhibitor.