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PubMed1995

Prevention of murine experimental allergic encephalomyelitis: cooperative effects of cyclosporine and 1 alpha, 25-(OH)2D3.

The hormone 1 alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3) has immune modulatory activities in vitro and in vivo, and can prevent or delay the onset of experimental or spontaneous autoimmune diseases. At therapeutical doses, however, hypercalcemic side effects are found. The present experiments examined the effects of combined treatment with subtherapeutic doses of cyclosporine A (CsA) and 1,25(OH)2D3 on the evolution of experimental autoimmune encephalomyelitis (EAE) in SJL mice. 1,25(OH)2D3 at 5 micrograms/kg body weight (given by i.p. injection every 2 days) prevented the appearance of paralysis in 70% of the treated mice. The treatment with 1,25(OH)2D3 at 2 micrograms/kg/2 days alone had less substantial protective effects (22% disease-free animals versus 5% in the control group). However, when this subtherapeutic dose was associated to treatment with a daily dose of CsA (2 or 5 mg/kg/day), which by itself was subtherapeutic (24 and 50% disease-free animals, respectively), the association of both drugs led to near-total protection (86% disease-free animals when combined with the highest dose of CsA). When an alternate day administration schedule (CsA at 10 mg/kg and 1,25(OH)2D3 at 2 micrograms/kg, each given on alternate days from day -3 to +19 after disease induction) was used, all treated mice were completely protected clinically and histologically. The two drugs also showed additive effects on serum osteocalcin and urinary calcium and desoxypyridinoline excretion, but not on serum calcium concentration. Our experiments demonstrate that 1,25(OH)2D3 might be a potential dose-reducing agent for CsA in immunosuppressive therapy.

Amino Acids↗

PubMed2000

Administration of IgG Fc fragments prevents glomerular injury in experimental immune complex nephritis.

Most human nephritis is due to glomerular deposition and/or formation of immune complexes (IC). In cultured mesangial cells, Fc receptor stimulation induces proliferation, matrix synthesis, and release of several mediators implicated in the initiation and progression of glomerular injury. Since Ig Fc fragments in vitro modified these phenomena, we studied the effects of systemic administration of IgG Fc fragments on the evolution of experimental IC nephritis. Fc fragment injection (1 mg/day i.p.) to rats with ongoing nephritis (proteinuria 20-50 mg/24 h vs 9 +/- 0.2 mg/24 h in controls) markedly ameliorates proteinuria, renal function, and morphological renal lesions. This was accompanied by a reduction in the renal synthesis of chemokines (monocyte chemoattractant protein-1, IFN-inducible protein-10, and cytokine-induced neutrophil chemoattractant-1), matrix proteins, and growth factors (platelet-derived growth factor, and TGF-beta), and in the activity of transcription factors. The treatment did not affect the glomerular deposition of IgG IC and complement C1q. In contrast, a decrease in the renal expression and production of C3 was observed without changes in serum complement levels. In vitro, very low complement consumption and no C3b covalent interaction were observed with Fc fragments, confirming that they did not modify systemic complement activity. These results indicate that the administration of Fc fragments prevents the development of glomerular damage in an aggressive model of proliferative glomerulonephritis through mechanisms involving a reduced local generation of complement, chemokines and growth factors. Modulation of IC-mesangial cell interaction by Fc fragment administration could represent a new approach to the treatment of severe immune nephritis.

Animals↗

PubMed1997

Comparison between three- and four-element Windkessel models to characterize vascular properties of pulmonary circulation.

In 11 anaesthetised pigs the accuracy of the three-element (WK3) and the four-element (WK4) Windkessel models to describe hemodynamic properties of the pulmonary circulation was compared during six different experimental conditions increasing pulmonary arterial pressure: increase in left atrial pressure, increase in alveolar pressure, increase in pulmonary blood flow, endotoxin shock, mechanical obstruction of left pulmonary artery or histamine infusion. Our results showed that WK4 fitted better the data than did WK3 because values of 1-R2 decreased from 6 percent (WK3) to 1.4 percent (WK4) when WK4 was used (P < 0.0005). 1-R2 was an adequate marker of the accuracy of the linear regression used to solve equations of both models. Compliance values estimated by WK4 were decreased by 5% comparatively to WK3 (P = 0.008). However, this difference can be considered as not physiologically relevant. Values of characteristic resistance corresponding to R1 + (L/R2C) in WK4 and to R1 in WK3 were not different (P = 0.22). The relative changes in R1, R2, and C observed due to the different experimental conditions were comparable regardless of the model. In conclusion, the conversion of WK3 in WK4 by adding an inductance, whose physiological meaning is not clear, resulted in an increased statistical accuracy of the model, but did not seem to have relevant influence on parameters or their evolution during experimental conditions.

Animals↗

PubMed1986

Experimental micropolycystic ovarian disease. I. Measurement of body weight, ovarian weight and histochemical activity of 17 beta-hydroxysteroid dehydrogenase.

The effects of administration of 1.25 mg testosterone propionate between the 2nd and 5th day of life on body weight, ovarian weight, food and water consumption, and the histochemical activity of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) were studied in female Wistar rats during the evolution of experimental micropolycystic ovaries. Biometric studies showed a 9.9 day delay of vaginal opening for the treated animals and that the increased body weight of the animals during the disease was not related to food intake and thus presumably due to the induced metabolic disorder. Reduced histochemical activity of the 17 beta-HSD of testosterone-treated rats was detected at ages 30, 60 and 90 days before the onset of morphological alterations of the ovaries. The possible participation of 17 beta-HSD in the disequilibrium between androstenedione and testosterone and in the genesis of the disease is discussed.

17-Hydroxysteroid Dehydrogenases↗

PubMed2002

The apparent clock-like evolution of Escherichia coli in glucose-limited chemostats is reproducible at large but not at small population sizes and can be explained with Monod kinetics.

To follow and model evolution of a microbial population in the chemostat, parameters are needed that give an indication of the absolute extent of evolution at a high resolution of time. In this study the evolution of the maximum specific growth rate ( micro (max)) and the residual glucose concentration was followed for populations of Escherichia coli K-12 under glucose-limited conditions at dilution rates of 0.1 x h(-1), 0.3 x h(-1) and 0.53 x h(-1) during 500-700 h in continuous culture. Whereas micro (max) improved only during the initial 150 h, the residual glucose concentration decreased constantly during 500 h of cultivation and therefore served as a convenient parameter to monitor the evolution of a population at a high time resolution with respect to its affinity for the growth-limiting substrate. The evolution of residual glucose concentrations was reproducible in independent chemostats with a population size of 10(11) cells, whereas no reproducibility was found in chemostats containing 10(7) cells. A model based on Monod kinetics assuming successive take-overs of mutants with improved kinetic parameters (primarily K(s)) was able to simulate the experimentally observed evolution of residual glucose concentrations. Similar values for the increase in glucose affinity of mutant phenotypes (K(s(mutant)) approximately equal 0.6 x K(s(parent))) and similar mutation rates per cell per generation leading to these mutant phenotypes (1-5 x1 0(-7)) were estimated in silico for all dilution rates. The model predicts a maximum rate of evolution at a dilution rate slightly below micro (max)/2. With increasing and decreasing dilution rates the evolution slows down, which also explains why in special cases a selection-driven evolution can exhibit apparent clock-like behaviour. The glucose affinity for WT cells was dependent on the dilution rate with highest values at dilution rates around micro (max)/2. Below 0.3 x h(-1) poorer affinity was mainly due to the effects of rpoS.

Adaptation, Physiological↗

PubMedDate not supplied

[Synaptic plasticity in learning and memory].

Pavlovian conditioning has been considered as one of the principal experimental approaches to understanding such complex brain functions as learning and memory. Use-dependent alterations in synaptic efficacy are believed to form the basis for these functions. The algorithm of synapse modification proposed by D. Hebb as early as 1949 is the coincident activation of pre- and postsynaptic neurons. The present review considers the evolution of experimental protocols which were used to reveal the manifestations of Hebb-type plasticity in the synaptic inputs to neocortical and hippocampal neurons. Special attention is focused on long-term modifications of synaptic efficacy in the hippocampus as a possible neuronal mechanism of learning and the role of disinhibition in their development. The effects of various neuromodulators on hippocampal long-term potentiation are considered. It is suggested that along with their involvement in disinhibition processes these substances may control the Hebb-type plasticity through intracellular second messenger systems.

Animals↗

PubMed1999

Comparison between MRI, microbiology and histology in evaluation of antibiotics in a murine model of thigh infection.

Although in vivo magnetic resonance imaging (MRI) is rapidly becoming a recognised tool in experimental pharmacological research, at the best of our knowledge, scarce application in the field of antibacterial drug research has been reported so far. In this last field, animal models of bacterial infections are used to test the efficacy of novel compounds. In this paper we have explored the potential usefulness of MRI in monitoring the chronological evolution of experimental bacterial infections and the effect of different therapeutic treatments. A murine model of thigh infection induced by Staphylococcus aureus has been used and the efficacy of vancomycin and imipenem/cilastatin has been tested. Three groups of infected animals were studied by microbiology, histology and MRI methods. The results obtained show that in vivo MRI data are highly consistent with microbiological and histological data, allowing, similarly to these commonly used techniques, the efficacy of different antibacterial treatments to be quantified. Our findings suggest that MRI could be used to assess the efficacy of new chemical entities in antibacterial pharmacological research. The advantages of MRI, as a non invasive technique, in comparison with commonly used microbiological and histological methods are discussed.

Animals↗

PubMed1988

Post-infectious ARDS: mechanisms of lung injury and repair.

Lung infections are considered the most common causes of ARDS. Additionally, superimposed secondary lung infections may occur in the setting of ARDS and further aggravate lung damage. Substances released by neutrophils are the major mediators of lung injury, although other factors, such as endotoxin, exotoxins, macrophages, and immune mechanisms, are significant contributors. Neutrophil-induced lung injury results from the production of oxygen radicals, the release of products of arachidonate metabolism, and the activation of lysosomal enzymes. The development of ARDS is associated with high mortality. Although a large proportion of survivors eventually recover adequate lung function, some patients progress to severe fibrosis. Experimental evidence suggests that the development of fibrosis results from increased synthesis of ECM components by lung fibroblasts. Fibroblast synthesis of ECM components is normally influenced by alveolar cells, by circulating growth factors, by the external milieu, and by the extent of gene expression of the specific ECM components. These factors undergo significant alterations during the evolution of experimental lung fibrosis. Growth factors are more abundant, alveolar cells are activated, and gene expression is enhanced. These factors, which play a crucial role in promoting experimental lung fibrosis, are likely to be involved in the pathogenesis of fibrosis in post-infectious ARDS.

Animals↗

PubMed2007

In silico panning for a non-competitive peptide inhibitor.

BACKGROUND: Peptide ligands have tremendous therapeutic potential as efficacious drugs. Currently, more than 40 peptides are available in the market for a drug. However, since costly and time-consuming synthesis procedures represent a problem for high-throughput screening, novel procedures to reduce the time and labor involved in screening peptide ligands are required. We propose the novel approach of 'in silico panning' which consists of a two-stage screening, involving affinity selection by docking simulation and evolution of the peptide ligand using genetic algorithms (GAs). In silico panning was successfully applied to the selection of peptide inhibitor for water-soluble quinoprotein glucose dehydrogenase (PQQGDH). RESULTS: The evolution of peptide ligands for a target enzyme was achieved by combining a docking simulation with evolution of the peptide ligand using genetic algorithms (GAs), which mimic Darwinian evolution. Designation of the target area as next to the substrate-binding site of the enzyme in the docking simulation enabled the selection of a non-competitive inhibitor. In all, four rounds of selection were carried out on the computer; the distribution of the docking energy decreased gradually for each generation and improvements in the docking energy were observed over the four rounds of selection. One of the top three selected peptides with the lowest docking energy, 'SERG' showed an inhibitory effect with Ki value of 20 microM. PQQGDH activity, in terms of the Vmax value, was 3-fold lower than that of the wild-type enzyme in the presence of this peptide. The mechanism of the SERG blockage of the enzyme was identified as non-competitive inhibition. We confirmed the specific binding of the peptide, and its equilibrium dissociation constant (KD) value was calculated as 60 microM by surface plasmon resonance (SPR) analysis. CONCLUSION: We demonstrate an effective methodology of in silico panning for the selection of a non-competitive peptide inhibitor from small virtual peptide library. This study is the first to demonstrate the usefulness of in silico evolution using experimental data. Our study highlights the usefulness of this strategy for structure-based screening of enzyme inhibitors.

Acinetobacter calcoaceticus↗

PubMed2006

Surfactant transfer through a liquid membrane: origin of spontaneous oscillations at the membrane/acceptor phase interface.

Instability due to surfactant redistribution in a liquid membrane system consisting of two solutions, namely source and acceptor, separated by a layer of immiscible liquid is studied theoretically and experimentally. The transfer of a surfactant from a source phase to an acceptor phase is often accompanied by spontaneous nonlinear oscillations of electrical potential and/or interfacial tension. The oscillations can be generated at each of the membrane interfaces. Here a mechanism of oscillation, which develops at the membrane/acceptor phase interface, is proposed on the basis of direct numerical simulation of the system evolution. Performed experimental studies confirm the theoretical results.

Alcohols↗

PubMed2006

Evolutionary influences in arboviral disease.

Arthropod-borne viruses (arboviruses) generally require horizontal transmission by arthropod vectors among vertebrate hosts for their natural maintenance. This requirement for alternate replication in disparate hosts places unusual evolutionary constraints on these viruses, which have probably limited the evolution of arboviruses to only a few families of RNA viruses (Togaviridae, Flaviviridae, Bunyaviridae, Rhabdoviridae, Reoviridae, and Orthomyxoviridae) and a single DNA virus. Phylogenetic studies have suggested the dominance of purifying selection in the evolution of arboviruses, consistent with constraints imposed by differing replication environments and requirements in arthropod and vertebrate hosts. Molecular genetic studies of alphaviruses and flaviviruses have also identified several mutations that effect differentially the replication in vertebrate and mosquito cells, consistent with the view that arboviruses must adopt compromise fitness characteristics for each host. More recently, evidence of positive selection has also been obtained from these studies. However, experimental model systems employing arthropod and vertebrate cell cultures have yielded conflicting conclusions on the effect of alternating host infections, with host specialization inconsistently resulting in fitness gains or losses in the bypassed host cells. Further studies using in vivo systems to study experimental arbovirus evolution are critical to understanding and predicting disease emergence, which often results from virus adaptation to new vectors or amplification hosts. Reverse genetic technologies that are now available for most arbovirus groups should be exploited to test assumptions and hypotheses derived from retrospective phylogenetic approaches.

Adaptation, Physiological↗

PubMed1989

The generation of transplasmic Drosophila simulans by cytoplasmic injection: effects of segregation and selection on the perpetuation of mitochondrial DNA heteroplasmy.

Experimental transplasmic Drosophila simulans were obtained through cytoplasm microinjection between eggs carrying different mitochondrial genomes. These genomes (siII and siIII) show a 1.5% difference in their sequences. They produced a large number of heteroplasmic flies in their F1 progeny and several flies were still heteroplasmic at the eighth generation. The distribution of frequencies of mitochondrial genotypes in the offspring of heteroplasmic females suggests that the stochastic processes involved in the evolution of experimental heteroplasmy of multiple nucleotide sites are very similar to those previously described for spontaneous length heteroplasmy. In addition, the siII genome has a noticeable advantage over the siIII genome in both directions of injection. This advantage is estimated at 58% per fly generation and 5% per cell generation.

Animals↗

PubMed2001

Computational method to reduce the search space for directed protein evolution.

We introduce a computational method to optimize the in vitro evolution of proteins. Simulating evolution with a simple model that statistically describes the fitness landscape, we find that beneficial mutations tend to occur at amino acid positions that are tolerant to substitutions, in the limit of small libraries and low mutation rates. We transform this observation into a design strategy by applying mean-field theory to a structure-based computational model to calculate each residue's structural tolerance. Thermostabilizing and activity-increasing mutations accumulated during the experimental directed evolution of subtilisin E and T4 lysozyme are strongly directed to sites identified by using this computational approach. This method can be used to predict positions where mutations are likely to lead to improvement of specific protein properties.

Bacteriophage T4↗

PubMed2003

Solution structure of a de novo protein from a designed combinatorial library.

Combinatorial libraries of de novo amino acid sequences can provide a rich source of diversity for the discovery of novel proteins. Randomly generated sequences, however, rarely fold into well ordered protein-like structures. To enhance the quality of a library, diversity must be focused into those regions of sequence space most likely to yield well folded structures. We have constructed focused libraries of de novo sequences by designing the binary pattern of polar and nonpolar amino acids to favor structures that contain abundant secondary structure, while simultaneously burying hydrophobic side chains in the protein interior and exposing hydrophilic side chains to solvent. Because binary patterning specifies only the polar/nonpolar periodicity, but not the identities of the side chains, detailed structural features, including packing interactions, cannot be designed a priori. Can binary patterned libraries nonetheless encode well folded proteins? An unambiguous answer to this question requires determination of a 3D structure. We used NMR spectroscopy to determine the structure of S-824, a novel protein from a recently constructed library of 102-residue sequences. This library is "naïve" in that it has not been subjected to high-throughput screens or directed evolution. The experimentally determined structure of S-824 is a four-helix bundle, as specified by the design. As dictated by the binary-code strategy, nonpolar side chains are buried in the protein interior, and polar side chains are exposed to solvent. The polypeptide backbone and buried side chains are well ordered, demonstrating that S-824 is not a molten globule and forms a unique structure. These results show that amino acid sequences that have neither been selected by evolution, nor designed by computer, nor isolated by high-throughput screening, can form native-like structures. These findings validate the binary-code strategy as an effective method for producing vast collections of well folded de novo proteins.

Amino Acid Sequence↗

PubMed2006

Matrix metalloproteinases, tissue inhibitors of MMPs and TACE in experimental cerebral malaria.

Cerebral malaria (CM) is a life-threatening disorder and a major medical problem in developing countries. It is caused by the sequestration of malaria-infected erythrocytes onto brain endothelia, followed by blood-brain barrier (BBB) damage and neurological deficit. In the present study, matrix metalloproteinases (MMPs) were analysed in a mouse model of CM with Plasmodium berghei ANKA. Increased numbers of gelatinase B (MMP-9)-positive cells, which were also CD11b(+), were detected in the brain. In addition, activation of gelatinase B occurred in CM brains, and not in brains of mice with non-CM. However, selective genetic knockout of gelatinase B did not alter the clinical evolution of experimental CM. To study other protease balances, the mRNA expression levels of nine matrix metalloproteinases (MMPs), five membrane-type MMPs, TNF-alpha converting enzyme (TACE) and the four tissue inhibitors of metalloproteinases (TIMPs) were analysed during CM in different organs. Significant alterations in expression were observed, including increases of the mRNAs of MMP-3, -8, -13 and -14 in the spleen, MMP-8, -12, -13 and -14 in the liver and MMP-8 and -13 in the brain. Net gelatinolytic activity, independent of gelatinase B and inhibitable with EDTA, was detected in situ in the endothelia of blood vessels in CM brains, but not in brains of mice with non-CM, suggesting that metalloproteases, different from gelatinase B, are active in the BBB environment in CM. The increase in MMP expression in the brain was significantly less pronounced after infection of C57Bl/6 mice with the noncerebral strain P. berghei NK65, but it was similar in CM-susceptible C57Bl/6 and CM-resistant Balb/C mice upon infection with P. berghei ANKA. Furthermore, in comparison with C57Bl/6 mice, a larger increase in TIMP-1 and a marked, >30-fold induction in MMP-3 were found in the brains of Balb/C mice, suggesting possible protective roles for TIMP-1 and MMP-3.

ADAM Proteins↗

PubMed2001

Loss of tubular bone morphogenetic protein-7 in diabetic nephropathy.

Bone morphogenetic protein-7 (BMP7), a member of the transforming growth factor-beta (TGF-beta) superfamily of cytokines, is highly expressed in renal tubules and generally promotes maintenance of epithelial phenotype. It was examined whether, during the evolution of experimental diabetic nephropathy, the renal expression of BMP7 and BMP7 receptors declines, and the hypothesis that loss of BMP7 activity is profibrogenic in proximal tubular cells was tested. Moreover, in vitro studies in cultured proximal tubular cells were performed to examine putative mechanisms that cause these changes. At 15 wk of streptozotocin-induced diabetes, renal expression of BMP7 is declined by about half, and it decreased further by 30 wk to <10% of timed controls. Renal expression of the high-affinity BMP type II receptor and the type I receptor Alk2 (activin receptor-like kinase-2) decreased. Alk3 tended to decrease, but Alk6 remained unchanged. During the evolution of diabetic nephropathy, the secreted BMP antagonist gremlin increased substantially. In cultured tubular cells, TGF-beta reduced BMP7 and Alk3 expression and increased gremlin but did not interrupt BMP7-induced activation of smad5 or Erk1 and -2. In contrast, BMP7 did not alter TGF-beta expression. Neutralization of endogenous BMP7 in cultured proximal tubular cells raised the expression of fibronectin and tended to increase collagen alpha(1) III mRNA levels. In conclusion, in experimental diabetic nephropathy, renal tubular BMP7 and some of its receptors decreased and gremlin, a secreted BMP antagonist, increased. Some, but not all, of these changes are explained by increased TGF-beta. The loss of BMP7 activity per se is profibrogenic in tubular cells.

Activins↗

PubMed1998

Isotachophoresis at pH extremes: theory and experimental validation.

The evolution of an isotachophoresis (ITP) system in acidic or basic pH ranges can be quite different from that predicted by the existing theory. It was found theoretically and proved experimentally that the contribution of hydrogen or hydroxyl ion to conductivity of solution and/or its net charge changes the behavior of the ITP system, creating in the terminating electrolyte an additional zone close to the initial interfaces between electrolytes (leader and terminator). One boundary of the zone, being either sharp or dispersed, moves toward the leader; the other is always sharp and stationary and coincides with initial electrolytes' discontinuity. The latter can be registered in the presence of electroosmotic flow which delivers it to the detection point. In order to describe the dynamics of the ITP system at pH extremes an algorithm of analytical solution was developed, based on the revised Kohlrausch theory. Its predictions coincide well with computer simulations and experimental data. The results presented can help in a correct analysis of ITP data and explain some confusing phenomena which were considered to be artifacts.

Algorithms↗

PubMed1986

Effects of skeletal change on muscle pattern formation.

The skeletal influence on muscle development was investigated taking an experimental and comparative approach. Chick hindlimbs of HH stage 23 were operated in order to induce the development of an enlarged, elongated fibula - similar to the condition in bird ancestors. The skeletal change resulted in a series of secondary alterations in the later-forming zeugopod muscles. By comparing the results to the zeugopod musculature in other birds and in reptiles it could be shown that changes did not occur at random but resembled the patterns of interspecific variation and ancestral arrangement. It is concluded that skeletal elements influence muscle morphogenesis during a certain period of development. A classification of four phases in muscle development is presented: formation of the premuscular masses; individuation of separate muscles; final shaping and insertion; development maintenance and growth. The factors acting on each level are discussed and it is suggested that skeletal influence becomes effective in phase 3. The results also emphasize the role of development in evolution. The experimental reestablishment of an ancestral epigenetic condition within a developing organ system resulted in ancestral features of subsequently forming characters. This suggests that modification of epigenetic control of gene expression may be an important mechanism in evolutionary change.

Alligators and Crocodiles↗