Rapid protocol for template preparation of large numbers of clones.
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Bacteriophages↗
Biomedical subjects
F Bartoli
Publications and source records attributed to F Bartoli.
Further characterization and comparison of inducible nitric oxide synthase in mouse, rat, and human hepatocytes.
Marked differences in induced nitric oxide (NO) synthesis occur between species. We have previously shown that both human and rat hepatocytes express an inducible NO synthase in response to cytokines and lipopolysaccharide. In this study, we compare the expression and regulation of cytokine-induced NO synthase in hepatocytes isolated from three species, human, rat, and mouse. On stimulation with tumor necrosis factor alpha (TNF alpha), interleukin-1 beta (IL-1 beta), interferon gamma (IFN gamma), and lipopolysaccharide (LPS), it was found that hepatocytes from all three species produce high levels of NO with levels of production exhibiting the following hierarchy: rat hepatocytes > mouse hepatocytes > human hepatocytes. Whereas rat and mouse hepatocytes express inducible NO synthase messenger RNA (mRNA) in response to TNF alpha, IL-1 beta, or IFN gamma as a single stimulus, human hepatocytes respond to LPS alone. Inhibition of NO generation through transforming growth factor (TGF-beta 1) was seen in mouse (77% +/- 5.9) and rat hepatocytes (17% +/- 2.6) whereas only about 10% was seen in human hepatocytes. Epidermal growth factor (EGF) was shown to inhibit NO synthesis in human and mouse hepatocytes but not rat. A marked NO-dependent inhibition of total protein synthesis was seen in rat and human hepatocytes, whereas mouse hepatocytes showed almost no inhibition in protein synthesis when stimulated. NO-dependent cyclic guanosine monophosphate (cGMP) release was found in all three species.(ABSTRACT TRUNCATED AT 250 WORDS)
1-Methyl-3-isobutylxanthine↗
Purification and partial characterization of draculin, the anticoagulant factor present in the saliva of vampire bats (Desmodus rotundus).
From the saliva of the vampire bat Desmodus rotundus, we isolated an unknown anticoagulant protein which we have named draculin. Its molecular mass as determined by non-reduced SDS-PAGE is about 83 kDa. The reduced polypeptide shows a slower migration. HPLC in a molecular sieve matrix yields a single, symmetrical peak corresponding to 88.5 kDa. Isoelectric focusing shows an acidic protein with pI = 4.1-4.2. Aminoacid analysis is compatible with a single chain polypeptide of about 80 kDa. Cyanogen bromide cleavage yields a single 16-aminoacid peptide, corresponding to the amino-terminus of the native molecule. Draculin inhibits the activated form of coagulation factors IX and X. It does not act on thrombin, trypsin, chymotrypsin and does not express fibrinolytic activity. The inhibition is immediate and not readily reversible, with a stoichiometry of about two molecules of draculin per molecule of factor IXa or Xa. Surprisingly, the inhibitory activity against either factor is not affected by the presence of the other. Draculin binds quantitatively to either immobilised factor Xa or factor IXa. Our preliminary interpretation is that there are two forms of draculin that hardly differ in structure. Both bind to factor Xa and to factor IXa but one form inhibits factor Xa and the other inhibits factor IXa. When added to plasma, draculin increases the lag phase as well as the height of the peak of thrombin generation.
Amino Acids↗
Tight binding inhibitors of 85-kDa phospholipase A2 but not 14-kDa phospholipase A2 inhibit release of free arachidonate in thrombin-stimulated human platelets.
An analogue of arachidonic acid in which the COOH group is replaced by a trifluoromethyl ketone group (COCF3) has recently been shown to be a tight binding inhibitor of the 85-kDa cytosolic phospholipase A2 that is found in platelets and other cells (Street, I. P., Lin, H.-K., Laliberté, F., Ghomashchi, F. G., Wang, Z., Perrier, H., Tremblay, N. M., Huang, Z., Weech, P. K., and Gelb, M. H. (1993) Biochemistry 32, 5935-5940). This trifluoromethyl ketone inhibits most of the arachidonate release from the phospholipid pool in thrombin-stimulated human platelets at concentrations of 0-40 microM with 4 x 10(8) platelets/ml. A structure-function analysis of related compounds reveals a good correlation between the inhibition of the purified phospholipase A2 and the blockage of arachidonate release in platelets. A number of recently described potent inhibitors of the 14-kDa phospholipase A2 that is secreted from activated platelets have no effect on the level of free arachidonate production. Furthermore, the addition of a large amount of recombinant 14-kDa phospholipase A2 to platelets does not produce free arachidonate, and it does not alter the amount of arachidonate released following platelet activation with thrombin. These studies provide strong pharmacological evidence for the role of the cytosolic phospholipase A2 in producing most, if not all, of the liberated arachidonate in thrombin-stimulated human platelets, and they show that tight binding membrane-residing inhibitors of the cytosolic phospholipase A2 can block the eicosanoid cascade in living cells.
Arachidonic Acids↗
Human hepatocytes are more resistant than rat hepatocytes to anoxia-reoxygenation injury.
We performed this study to determine whether perfused isolated human and rat hepatocytes have different sensitivities to anoxia-reoxygenation injury. Oxygen free radicals were detected by lucigenin-enhanced chemiluminescence. Lipid peroxidation was assessed by measuring malondialdehyde release. Cell injury was evaluated by measuring lactate dehydrogenase release and trypan blue uptake. During the control period, lucigenin-enhanced chemiluminescence, malondialdehyde and lactate dehydrogenase release and trypan blue uptake were similar in rat and human hepatocytes. During 3.5 hr of anoxia, lucigenin-enhanced chemiluminescence decreased to background levels and malondialdehyde release remained constant in both groups. In contrast, lactate dehydrogenase release increased eightfold in rat hepatocytes but only threefold in human hepatocytes. With reoxygenation after 2.5 hr of anoxia, in rat hepatocytes lucigenin-enhanced chemiluminescence increased 13-fold within 15 min and then declined toward control levels. Malondialdehyde release doubled after 1 hr of reoxygenation. The rate of lactate dehydrogenase release increased to a level almost twice that observed in cells kept continuously anoxic. In contrast, with human hepatocytes lucigenin-enhanced chemiluminescence increased only fourfold, whereas malondialdehyde and lactate dehydrogenase releases did not differ significantly from those levels measured in cells perfused continuously under anoxic conditions. At the end of the experiment, the increase in trypan blue uptake was significantly greater with rat hepatocytes than with human hepatocytes. These results demonstrate that (a) during reoxygenation following 2.5 hr of anoxia, isolated human hepatocytes generate fewer oxygen free radical, and lipoperoxides than do rat hepatocytes, and (b) human hepatocytes are more resistant to cell injury during anoxia-reoxygenation than are rat hepatocytes.
Adult↗
Human hepatocytes differ from rat hepatocytes in their sensitivity to anoxia-reoxygenation injury.
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Aerobiosis↗
Hemodynamic changes during acute rejection of small bowel allografts.
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Analysis of Variance↗
Evidence for direct coupling of primary agonist-receptor interaction to the exposure of functional IIb-IIIa complexes in human blood platelets. Results from studies with the antiplatelet compound ajoene.
Ajoene, (E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide, is a potent antiplatelet compound isolated from alcoholic extracts of garlic. In vitro, ajoene reversibly inhibits platelet aggregation as well as the release reaction induced by all known agonists. In this paper we show that ajoene has a unique locus of action, that is not shared by any other known antiplatelet compound. For example, ajoene inhibits agonist-induced exposure of fibrinogen receptors, as well as intracellular responses such as activation of protein kinase C and the increase in cytoplasmic free calcium induced by receptor-dependent agonists (collagen, ADP, PAF, low-dose thrombin). On the other hand, with agonists that can by-pass (at least partially) the receptor-transductor-effector sequence, such as high-dose thrombin, PMA, NaF, only the exposure of fibrinogen receptors is blocked by ajoene. Binding of fibrinogen to chymotrypsin-treated platelets is only slightly inhibited by ajoene. The results reported here also show that: (a) ajoene does not act as a calcium chelator, does not impair the initial agonist-receptor interaction and does not influence the basal levels of intracellular inhibitors of platelet activation such as cyclic GMP; (b) the locus of action of ajoene is a yet unknown molecular step that links, in the case of physiological agonists, specific agonist-receptor complexes to the sequence of the signal transduction system on the plasma membrane of platelets. In the case of non-physiological, receptor-independent agonists (PMA, NaF), we can only speculate on the hypothesis that they somehow mimic the effect of the agonist-receptor complexes on the signal transduction system; and (c) the exposure of fibrinogen receptors is not a direct consequence of other intracellular processes. These observations clearly show, for the first time, that the exposure of fibrinogen receptors is a membrane event proximally and obligatorily coupled to the occupancy of other membrane receptors by their agonists without any intervention by the cytoplasmic biochemical processes. Additional results support the involvement of G-proteins in these early events of platelet activation. Furthermore, a role of the beta tau subunits of G-proteins in the exposure of fibrinogen receptors is proposed.
Antibodies, Monoclonal↗
The effects of long-term hypoxia on epicardium and myocardium in developing chick embryo hearts.
The consequences of long-term O2 deprivation on heart development were analyzed morphometrically and ultrastructurally, utilizing the hearts of chicken embryos developed under hypoxia from the 3rd to the 18th incubation day. The results indicate that embryos kept under low O2 blood tension do not show disturbances in heart morphohistogenesis, but are characterized by a thicker epicardium and a thinner myocardium than the controls; moreover, both the number and calibre of the heart microvessels are increased. The thickening of the epicardium is due to hyperplasia of the mesothelial cells, increment in calibre of the submesothelial vessels, and to conspicuous perivascular infiltration of blood-derived cells. The thinning of the cardiac muscle seems to be dependent on myocardiocyte hypotrophy and myofibril reduction. The increase in the volume density of myocardium vessels, due to their dilatation and proliferation, may be considered expression of a vascular adaptive reaction to low oxygen tissue concentration.
Animals↗
Intestinal ischemia: morphological features--II.
Superoxide radicals produced during acute intestinal ischemia are biochemically related with the presence of hydrogen peroxyde. In this study we have investigated the distribution of peroxidase-catalase activity, histochemically determined, in the ischemic ileal wall. In the rat, complete arterial and venous occlusion produced a progressive increase in extra-vascular peroxidase-catalase activity with a maximum corresponding to the ileal wall. Probably the tissue peroxidase-catalase activity is related to massive degranulation of polymorphonucleates.
Animals↗
Intestinal occlusion: morphological features.
In this research we studied the possible relationships between the level and the distribution of peroxidase-catalase activity and the degree of the morphological changes in the intestinal wall during mechanical occlusion. These researches have really proved that the increase of the peroxidase-catalase enzymes correlate proportionally with the damage of intestinal wall.
Animals↗
[Blood levels of vitamin E, polyunsaturated fatty acids of phospholipids, lipoperoxides, glutathione peroxidase activity and serological screening for syphilis and HIV in immigrants from developing countries].
Plasma levels of vitamin E (Vit E), polyunsaturated fatty acids of phospholipids (PUFA-PL), lipoperoxides as well as erythrocytes glutathione peroxidase activity (GSH-Px), were evaluated in 200 migrants coming from developing countries, some of which at high risk for serious infective diseases. HIV-1 and syphilis infections were also investigated. 114 subjects (57%) had blood levels of Vit E, PUFA-PL and GSH-Px significantly lower (p less than 0.001, p less than 0.01) than those of normal healthy individuals (n = 30), while lipoperoxides values were unchanged. 8 from this group were found to be HIV-1 positive, and 5 TPHA positive. In contrast, the remaining 86 migrants did not show any signs of infections and their blood parameters were normal enough. These results show that factors such as widespread poverty, inadequate housing, malnutrition, insufficient access to medical care, psychological stress are strictly correlated to the reduction of blood parameters which are critical for the normal cell function of mammalian cells. PUFA-PL deficiency may cause lesions likely due to faulty cellular membranes. The lack of Vit E and GSH-Px, which are considered major protective molecules against lipoperoxidation damage in vivo, has been involved in several human diseases. We suggest that low blood levels of Vit E, GSH-Px and particularly PUFA-PL may play a pathogenetic role in the onset and development of AIDS and other infections. In this connection, we have found that a deficiency of these blood parameters occurs in patients with AIDS (n = 50) and in 32% of HIV seronegative intravenous drug abusers (n = 100).
Acquired Immunodeficiency Syndrome↗
Intestinal ischemia: morphological features--I.
In this research we studied the interrelationships between the progression of morphological damage and leucocyte populations in the last ileal loop after blockage of blood supply subsequent to arterial and venous occlusion. Our morphological data on the staging of the ileal wall damage agree with those reported in literature. In addition we described that in the ischemic loop the number of lymphocytes appear decreased and polymorphonuclear cells degranulate actively.
Animals↗
[Histologic study of the pyelo-ureteral junction].
The ureter structure was analyzed under light microscope on serial sections in newborn children affected by obstruction of the pyelo-ureteric junction. In the obstructive segments, preceded by ureter portions dilated and provided with close-packed layers of smooth muscle layers, the tunica mucosa was lacking epithelial cover, its lamina propria was thickened, being built by conspicuous bundles of collagen fibers, and the tunica muscularis showed scarce and disrupted groups of muscle cells invaded by connective tissue. Numerous mastocytes were seen in the mucosa and muscularis tunicae. The results suggest that the breaking of the epithelium may be a primary pathogenetic event followed by passage of urine in the subjacent tissues in turn responsible for a diffuse connective reaction, and, therefore for a final fibrosis of the ureter wall. The role of the mastocytes in the etiopathogenesis of the pyelo-ureteric junction obstruction was also discussed.
Constriction, Pathologic↗
In vivo platelet hyperreactivity, another risk factor for patients under continuous ambulatory peritoneal dialysis.
A high mortality rate due to thromboembolic accidents has been described in patients undergoing chronic haemodialysis. This type of complications, although recognized, has not been appropriately evaluated in continuous ambulatory peritoneal dialysis patients. The present study demonstrates that continuous ambulatory peritoneal dialysis patients present in vivo platelet hyperreactivity, as evidenced by enhanced platelet responses to epinephrine ex vivo and an increased MDA/MDAa index which traduces a decreased threshold for activation of the arachidonate pathway and subsequent thromboxane production. Since the etiopathogeny of this platelet abnormality seems to be related to abnormalities in lipid metabolism, compounds such as fish oil must be beneficial in the management of this risk factor.
Adult↗
[Effects of chronic hypoxia on erythrocytes and plasma components of blood during development of the chick embryo].
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Animals↗
AR identification and spectral estimate applied to the R-R interval measurements.
The methods of identification and spectral estimate are applied to the tachogram, i.e. the time series constituted by the cycle-by-cycle R-R interval durations measured on the ECG signal from cardiological patients in ambulatory rehabilitation training after episodes of myocardial infarction or ischemic disease. The Batch Least Squares Method is applied to identify the series as an AR process of 5th order. The whiteness test and Rissanen's optimization criterion are also fulfilled. The clinical information is in this way highly compressed in the pole diagram and in the Maximum Entropy Spectrum (MES) estimated on the basis of the AR coefficients. The experimental results in a restricted set of patients confirm the feasibility of new instrumentation design criteria for non-conventional R-R intervals parametrisation, successive diagnostic classification and beat prediction. Finally, some preliminary considerations about the capabilities of the introduced methods put into evidence the role of computerized techniques in recognizing the fundamental patterns of physiopathological heart rate variability, which the usual conventional methods of ECG analysis are not able to detect in a reliable way.
Arrhythmias, Cardiac↗
An optimal linear filter for the reduction of noise superimposed to the EEG signal.
In the present paper a procedure for the reduction of super-imposed noise on EEG tracings is described, which makes use of linear digital filtering and identification methods. In particular, an optimal filter (a Kalman filter) has been developed which is intended to capture the disturbances of the electromyographic noise on the basis of an a priori modelling which considers a series of impulses with a temporal occurrence according to a Poisson distribution as a noise generating mechanism. The experimental results refer to the EEG tracings recorded from 20 patients in normal resting conditions: the procedure consists of a preprocessing phase (which uses also a low-pass FIR digital filter), followed by the implementation of the identification and the Kalman filter. The performance of the filters is satisfactory also from the clinical standpoint, obtaining a marked reduction of noise without distorting the useful information contained in the signal. Furthermore, when using the introduced method, the EEG signal generating mechanism is accordingly parametrized as AR/ARMA models, thus obtaining an extremely sensitive feature extraction with interesting and not yet completely studied pathophysiological meanings. The above procedure may find a general application in the field of noise reduction and the better enhancement of information contained in the wide set of biological signals.
Computers↗