High-Frequency Linear Array (20 MHz) Based on Lead-Free BCTZ Crystal.

Centimeter-sized BaTiO3-based crystals grown by top-seeded solution growth from the BaTiO3-CaTiO3-BaZrO3 system were used to process a high-frequency (HF) lead-free linear array. Piezoelectric plates with (110)pc cut within 1° accuracy were used to manufacture two 1-3 piezo-composites with thicknesses of 270 and [Formula: see text] for resonant frequencies in air of 10 and 30 MHz, respectively. The electromechanical characterization of the BCTZ crystal plates and the 10-MHz piezocomposite yielded the thickness coupling factors of 40% and 50%, respectively. We quantified the electromechanical performance of the second piezocomposite (30 MHz) according to the reduction in the pillar sizes during the fabrication process. The dimensions of the piezocomposite at 30 MHz were sufficient for a 128-element array with a 70- [Formula: see text] element pitch and a 1.5-mm elevation aperture. The transducer stack (backing, matching layers, lens, and electrical components) was tuned with the characteristics of the lead-free materials to deliver optimal bandwidth and sensitivity. The probe was connected to a real-time HF 128-channel echographic system for acoustic characterization (electroacoustic response and radiation pattern) and to acquire high-resolution in vivo images of human skin. The center frequency of the experimental probe was 20 MHz, and the fractional bandwidth at -6 dB was 41%. Skin images were compared against those obtained with a lead-based 20-MHz commercial imaging probe. Despite significant differences in sensitivity between elements, in vivo images obtained with a BCTZ-based probe convincingly demonstrated the potential of integrating this piezoelectric material in an imaging probe.

Anisotropic mechanical characterization of human skin by in vivo multi-axial ring suction test.

Human skin is a soft tissue behaving as an anisotropic material. The anisotropy emerges from the alignment of collagen fibers in the dermis, which causes the skin to exhibit greater stiffness in a certain direction, known as Langer's line. The importance of determining this anisotropy axis lies in assisting surgeons in making incisions that do not produce undesirable scars. In this paper, we introduce an open-source numerical framework, MARSAC (Multi-Axial Ring Suction for Anisotropy Characterization: https://github.com/aflahelouneg/MARSAC), adapted to a commercial device CutiScan CS 100® that applies a suction load on an annular section, causing a multi-axial stretch in the central zone, where in-plane displacements are captured by a camera. The presented framework receives inputs from a video file and converts them into displacement fields through Digital Image Correlation (DIC) technique. From the latter and based on an analytical model, the method assesses the anisotropic material parameters of human skin: Langer's line ϕ, and the elastic moduli E1 and E2 along the principal axes, providing that the Poisson's ratio is fixed. The pipeline was applied to a public data repository, https://search-data.ubfc.fr/femto/FR-18008901306731-2021-08-25_In-vivo-skin-anisotropy-dataset-for-a-young-man.html, containing 30 test series performed on a forearm of a Caucasian subject. As a result, the identified parameter averages, ϕˆ=40.9±8.2∘ and the anisotropy ratio E1ˆ/E2ˆ=3.14±1.60, were in accordance with the literature. The intra-subject analysis showed a reliable assessment of ϕ and E2. As skin anisotropy varies from site to site and from subject to subject, the novelty of the method consists in (i) an optimal utilization of CutiScan CS 100® probe to measure the Langer's line accurately and rapidly on small areas with a minimum diameter of 14mm, (ii) validation of an analytical model based on deformation ellipticity.

Evaluation of ponatinib in vitro effect in three canine mast cell tumor cell lines expressing FGFR-1, PDGFR-α, and VEGFR-2.

Ponatinib is a broad-spectrum tyrosine kinase inhibitor that targets numerous receptor tyrosine kinases (RTKs), including but not limited to fibroblast growth factor receptor (FGFR)-1, platelet derived growth factor receptor (PDGFR)-α, and vascular endothelial growth factor receptor (VEGFR)-2. This study evaluated the expression of FGFR-1, PDGFR-α, and VEGFR-2 in three canine mast cell tumor (MCT) cell lines (CM-MC1, VI-MC1, CoMS) and the effects of ponatinib on these MCT cell lines. Quantitative RT-PCR confirmed the expression of FGFR-1, PDGFR-α, and VEGFR-2 in the three MCT cell lines. Ponatinib exhibited dose- and time-dependent cytotoxicity in MCT cell lines via MTT assay. The IC50 for 24, 48, and 72 h across the three cell lines ranged from 38.47 nM to 103.3 nM, which is clinically comparable to dose ranges established for humans. Significantly increased apoptosis in each cell line was seen between 12 and 18 h after treatment with IC50 of ponatinib via Annexin-V and Caspase-3/7 assays. These data suggest that ponatinib could be a possible therapeutic agent for canine MCTs. Further studies are needed to investigate the prognostic value of FGFR-1, PDGFR-α, and VEGFR-2 in canine MCTs.

Analysis of canine myeloid-derived suppressor cells (MDSCs) utilizing fluorescence-activated cell sorting, RNA protection mediums to yield quality RNA for single-cell RNA sequencing.

Fluorescence-activated cell sorting (FACS) is a branch of flow cytometry that allows for the isolation of specific cell populations that can then be further analyzed by single-cell RNA sequencing (scRNA-seq). When utilizing FACS for population isolation prior to sequencing, it is essential to consider the protection of RNA from RNase activity, environmental conditions, and the sorting efficiency to ensure optimum sample quality. This study aimed to optimize a previously published MDSC flow cytometry strategy to FACS sort canine Myeloid-Derived Suppressor Cells (MDSC) with various permutations of RNAlater ™ and RiboLock™ before and after FACS sorting. Concentrations of RNAlater™ greater than 2 % applied before flow analysis affected cell survival and fluorescence, whereas concentrations ≤ 2 % and time ≤ 4 h had little to no effect on cells. To shorten the procedural time and to enhance the sorting of rare populations, we used a primary PE-conjugated CD11b antibody and magnetic column. The combination of RiboLock™ pre- and post-sorting for FACS provided the best quality RNA as determined by the RNA integrity number (RIN ≥ 7) for scRNA-seq in a normal and dog and a dog with untreated oral melanoma dog. As proof of principle, we sequenced two samples, one from a normal dog another from a dog with untreated oral melanoma. Applying scRNA-Seq analysis using the 10X Genomic platform, we identified 6 clusters in the Seurat paired analysis of MDSC sorted samples. Two clusters, with the majority of the cells coming from the melanoma sample, had genes that were upregulated (> log2); these included MMP9, MMP1, HPGD, CPA3, and GATA3 and CYBB, CSTB, COX2, ATP6, and COX 17 for cluster 5 and 6 respectively. All genes have known associations with MDSCs. Further characterization using pathway analysis tools was not attempted due to the lower number of cells sequenced in the normal sample. The benefit deriving from the results of the study helped to gain data consistency when working with cells prone to RNase activity, and the scRNA-seq provided data showing transcriptional heterogeneity in MDSC populations and potentially identifying previously unreported or rare cell populations.

An open source pipeline for design of experiments for hyperelastic models of the skin with applications to keloids.

The aim of this work is to characterize the mechanical parameters governing the in-plane behavior of human skin and, in particular, of a keloid-scar. We consider 2D hyperelastic bi-material model of a keloid and the surrounding healthy skin. The problem of finding the optimal model parameters that minimize the misfit between the model observations and the in vivo experimental measurements is solved using our in-house developed inverse solver that is based on the FEniCS finite element computational platform. The paper focuses on the model parameter sensitivity quantification with respect to the experimental measurements, such as the displacement field and reaction force measurements. The developed tools quantify the significance of different measurements on different model parameters and, in turn, give insight into a given model's ability to capture experimental measurements. Finally, an a priori estimate for the model parameter sensitivity is proposed that is independent of the actual measurements and that is defined in the whole computational domain. This estimate is primarily useful for the design of experiments, specifically, in localizing the optimal displacement field measurement sites for the maximum impact on model parameter inference.

On the impact of ethanol on the rejection and transfer mechanism during ultrafiltration of a charged macromolecule in water/ethanol.

Ultrafiltration (UF) is a sustainable membrane separation technique. It could be useful for the concentration/purification of bio-sourced molecules that are extracted either by pure ethanol or by water/ethanol mixtures. Nevertheless, the process optimization requires an in-depth understanding of the transfer mechanisms of solute through membranes, especially for charged solutes, that are nowadays not sufficiently documented. Previous studies achieved in aqueous media have shown that the rejection of charged solutes by an UF membrane involves at least three mechanisms: convection, diffusion and electrostatic interactions. The present study aims at a systematic analysis of the transfer mechanisms of a model protein (lysozyme) in water/ethanol mixtures (100/0-70/30 v/v) during UF by a zirconia inorganic membrane. The influence of the pH varying in the 4-9 range and of the ionic strength (I) is also discussed. The ionic strength I can be adjusted by addition of an indifferent electrolyte (NaCl) only aiming at the screening of the electrostatic interactions or by addition of a selectively adsorbed electrolyte(KH2PO4) that is able to change the isoelectric pH of the protein and thus to modulate the electrostatic interactions in a different way when compared to NaCl. Of course, both salts have an impact on the protein rejection in UF. The results are analysed using the CDE model previously developed in our group to explain the behaviour of a single protein during UF in water and accounting for convection, diffusion and electrophoretic migration. The applicability of the CDE model in water/ethanol mixtures up to 70/30 v/v is finally shown.

Characterization of myeloid-derived suppressor cells and cytokines GM-CSF, IL-10 and MCP-1 in dogs with malignant melanoma receiving a GD3-based immunotherapy.

Melanoma in humans and canines is an aggressive and highly metastatic cancer. The mucosal forms in both species share genetic and histopathologic features, making dogs a valuable spontaneous disease animal model. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells of myeloid origin with immunosuppressive capabilities, which are increased in many human cancers and contribute to tumor immune evasion. They are a possible target to improve immunotherapy outcomes. Current information regarding MDSCs in canines is minimal, limiting their use as translational model for the study of MDSCs. The objective of this study was to characterize major MDSCs subsets (monocytic and polymorphonuclear) and the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 10 (IL-10) and monocyte chemoattractant protein-1 (MCP-1) in canines with malignant melanoma and to evaluate changes in MDSCs and the cytokines over time in response to a GD3-based active immunotherapy. Whole blood and serum collected from 30 healthy controls and 33 patients enrolled in the University of Florida melanoma vaccine trial were analyzed by flow cytometry with canine specific CD11b, MHCII and anti-human CD14 antibodies to assess ostensibly polymorphonuclear-MDSC (CD11b+ MHCII- CD14-) and monocytic-MDSC (CD11b+ MHCII- CD14+) subsets. IL-10, MCP-1 and both MDSCs subsets were significantly elevated in melanoma dogs versus controls. Both MDSCs subsets decreased significantly following GD3-based immunotherapy administration but no significant changes in cytokines were seen over time. To our knowledge, this is the first report documenting increased monocytic-MDSCs in canine melanoma. This is consistent with human malignant melanoma data, supporting dogs as a valuable model for therapeutic intervention studies.

Response rate to a single dose of vinblastine administered to dogs with treatment-naive multicentric lymphoma.

Vincristine is included in vincristine, cyclophosphamide, doxorubicin and prednisone (CHOP) chemotherapy protocols, which are the gold-standard treatment for high-grade canine lymphoma. Vincristine can result in relatively high rates of gastrointestinal toxicity, whereas vinblastine is generally well tolerated and thus may represent an under-utilized and minimally toxic alternative to vincristine. Our objective was to determine the response rate and toxicity associated with a single dose of vinblastine administered to dogs with treatment-naïve, intermediate to large-cell, multicentric lymphoma. Twenty client-owned dogs were enrolled with signed owner consent. A Simon's minimax, phase II, two-stage trial was performed to test the efficacy of vinblastine administered at 2 mg/m2 IV followed by a pilot trial of vinblastine at 2.5 mg/m2 . No dogs were administered concurrent steroids or other chemotherapy. One out of 14 dogs receiving vinblastine at 2 mg/m2 demonstrated a partial response. Three out of five dogs demonstrated a partial response to vinblastine at 2.5 mg/m2 . Gastrointestinal toxicity was infrequent and low grade for both groups. The majority of dogs (80%) in the 2.5 mg/m2 dosing group developed neutropenia 1-week post administration. Vinblastine was well tolerated but minimally efficacious at a dose of 2 mg/m2 IV in dogs with treatment-naive, multicentric lymphoma. Because of poor response rates, treatment at this dose is not recommended. A small subset of dogs administered 2.5 mg/m2 had significantly improved response rates (P = 0.04), suggesting that higher doses may have improved efficacy, although further research is indicated to confirm these preliminary findings.

Electric field induced Lyman-α emission of a hydrogen beam for electric field measurements.

Electric field induced Lyman-α emission is a new way of measuring weak electric fields in vacuum and in a plasma. It is based on the emission of Lyman-α radiation (121.6 nm) by a low-energy metastable H atom beam due to Stark-quenching of the 2s level induced by the field. In this paper, we describe the technique in detail. Test measurements have been performed in vacuum between two plates polarized at a controlled voltage. The intensity of emitted radiation, proportional to the square of the field modulus, has been recorded by a lock-in technique, which gives an excellent signal to noise ratio. These measurements provide an in situ calibration that can be used to obtain the absolute value of the electric field. A diagnostic of this type can help to address a long standing challenge in plasma physics, namely, the problem of measuring electric fields without disturbing the equilibrium of the system that is being studied.

Aggressive local therapy combined with systemic chemotherapy provides long-term control in grade II stage 2 canine mast cell tumour: 21 cases (1999-2012).

This retrospective case series evaluates the outcome of 21 dogs with grade II stage 2 mast cell tumour (MCT) treated with adequate local therapy and adjuvant systemic chemotherapy (prednisone, vinblastine and CCNU). The median survival for all dogs was 1359 days (range, 188-2340). Median disease-free interval was 2120 days (149-2325 days). Dogs treated with surgery and chemotherapy had shorter survival (median, 1103 days; 188-2010 days) than those that underwent surgery, radiation therapy and chemotherapy as part of their treatment (median, 2056 days; 300-2340 days). Two patients had local recurrence in the radiation field and four patients had de novo MCT. Distant metastasis was not observed in any dogs. The results of this study suggest that, in the presence of loco-regional lymph node metastasis in grade II MCT, the use of prednisone, vinblastine and CCNU after adequate local-regional therapy can provide a median survival in excess of 40 months.

Predictive and concurrent validity of standardized neurodevelopmental examinations by the Griffiths scales and Bayley scales of infant development II.

Standardized examinations of preterm infants are used to identify candidates for early intervention. We aimed to assess the predictive power and concurrent validity of the mental development index of the Bayley scales of infant development II (Bayley MDI) and the Griffiths scales developmental quotient (Griffiths DQ) in healthy term and preterm infants <1500 g birth weight without major perinatal complications.137 Infants (89 term, 48 preterm) were examined by both tests at a corrected age of 6, 12, and 22 months, and 114 went on to undergo Bayley assessments at 39 months.There were significant correlations between Bayley and Griffiths results at 6, 12, and 22 months (r=0.530, 0.714, and 0.833, respectively, p<0.001) but Bland Altman plots revealed major systematic bias at 6 months (Griffiths>Bayley, mean differences 14.3±9.8) and 22 months (Bayley>Griffiths, mean difference 5.2±13.9) and wide 95% limits of agreement at 6, 12 and 22 months (35.9%, 40.0%, and 52.4%, respectively). The agreement for a presumptive diagnosis of developmental impairment in the group of preterm infants between Bayley examinations obtained at 39 months corrected age (reference) and previous examinations was poor at 6, 12, and 22 months for both Bayley and Griffiths (Cohen's kappa for Griffiths: 0.225, 0.192, 0.369; for Bayley: 0.121, 0.316, 0.369, respectively).Caution should be exercised when interpreting results from standardized neurodevelopmental examinations obtained during the first 2 years of life in comparatively well preterm infants.

IL-10 and TGF-beta1 are associated with variations in fluke burdens following experimental fasciolosis in sheep.

Infection with Fasciola hepatica causes an economically important disease in ruminants. Variability in parasite load may indicate innate differences in the host immune system. This study aimed to investigate the immunological mechanisms that are associated with variability in parasite burden following experimental F. hepatica infection in cross-bred sheep. Of a total of 16 animals, four were randomly chosen as uninfected controls, and the remainder infected with 100 viable metacercariae. Uninfected animals were used as the control group for evaluation of cytokine gene expression levels. For comparative analysis, specific animals were selected on the basis of extremes of fluke burdens, and were categorised into light (n = 4) and heavy burdened (n = 3) cohorts. Serum antibody levels, haematological parameters, and expression of IL-4 and IFN-gamma genes in hepatic lymph nodes were equivalent in both groups. However, significant differences in mitogen-specific lymphocyte proliferation in vitro and in expression of TGF-beta1 and IL-10 genes in hepatic lymph nodes were observed at acute and chronic phases of infection, respectively. These results provide useful information in developing further understanding of natural resistance to fasciolosis in sheep.

Stochastic models to study the impact of mixing on a fed-batch culture of Saccharomyces cerevisiae.

The mechanisms of interaction between microorganisms and their environment in a stirred bioreactor can be modeled by a stochastic approach. The procedure comprises two submodels: a classical stochastic model for the microbial cell circulation and a Markov chain model for the concentration gradient calculus. The advantage lies in the fact that the core of each submodel, i.e., the transition matrix (which contains the probabilities to shift from a perfectly mixed compartment to another in the bioreactor representation), is identical for the two cases. That means that both the particle circulation and fluid mixing process can be analyzed by use of the same modeling basis. This assumption has been validated by performing inert tracer (NaCl) and stained yeast cells dispersion experiments that have shown good agreement with simulation results. The stochastic model has been used to define a characteristic concentration profile experienced by the microorganisms during a fermentation test performed in a scale-down reactor. The concentration profiles obtained in this way can explain the scale-down effect in the case of a Saccharomyces cerevisiae fed-batch process. The simulation results are analyzed in order to give some explanations about the effect of the substrate fluctuation dynamics on S. cerevisiae.

Thermodynamics from three-dimensional many-body fragmentation simulations on a cellular automaton model.

The thermal equilibrium of many-body systems subject to finite range interactions is investigated numerically, by means of a multipurpose 3D cellular automaton dynamic model developed by the authors. The numerical experiments, carried out at fixed number of bodies, volume and energy, demonstrate the formation of an equilibrium among 3D aggregates of bodies. The distribution of the aggregates against size obeys a power law of (negative) exponent tau approximately 2.2 (against 1.3 in 2D). Our experiments, indicating that the exponent is insensitive to the precise parameter values and the precise parametrization of the interactions, are consistent with the idea of the existence of a universality class corresponding to the thermal equilibrium. The numerical value for the exponent tau is in agreement with the theoretical thermal equilibrium analyses based on various other approaches, numerical and semianalytical, indicating that the cellular automaton approach provides an adequate methodology to investigate thermal equilibria. In this paper, as an illustration of this method, we refer to the problem of formation of clusters of nucleons in heavy ion collisions of nuclei leading on to fragmentation. The theoretical tau value, however, corresponding to the thermal equilibrium among the aggregation clusters, is 15 percent lower than the empirical value ( approximately 2.6 ) , as measured in laboratory nuclear fragmentation experiments induced by collision. There is then only a very approximate correspondence between the experimental and the thermal equilibrium value. On the basis of the results of this paper and of a previous paper of this series, we conjecture that the approximate agreement is due to a partial establishment of a thermodynamic equilibrium during the collision of the nuclei. The thermal equilibrium gives the main contribution to the observed tau value; the deviation from this possibly universal value is largely the consequence of the lack of full thermal equilibrium in actual laboratory experiments. This conjecture is extended to interpret the observed ubiquity of power laws of exponents exceeding 2.2, which refer to the distribution of various types of matter in 3D space.

Dynamics of many-particle fragmentation in a cellular automaton model.

A three-dimensional cellular automaton model developed by the authors to deal with the dynamics of N-body interactions has been adapted to investigate the head-on collision of two identical bound clusters of particles, and the ensuing process of fragmentation. The range of impact energies is chosen low enough, to secure that a compound bound cluster can be formed. The model is devised to simulate the laboratory set-up of fragmentation experiments as monitored by 4 pi detectors. The particles interact via a Lennard-Jones potential. At low impact energies the numerical experiments following the dynamics of the individual particles indicate a phase of energy sharing among all the particles of the compound cluster. Fragments of all sizes are then found to evaporate from the latter cluster. The cluster sizes, measured in our setup by simulated 4 pi detectors, conform to a power law of exponent approximately 2.6. In an attempt to duplicate the laboratory caloric curves related, in particular, to nuclear fragmentation processes, we introduce several temperature parameters (kinetic temperature of nucleons, kinetic temperature of fragments, reaction equilibrium temperatures). Theoretical caloric curves are then constructed for those temperature parameters, we regard as physically most relevant. Our results show that different temperature definitions generate different curve patterns, indicating that the fragmentation system remains far from thermodynamic equilibrium. The pattern of the laboratory caloric curve for Au-Au collision experiments as derived from a recent analysis [NuPECC Report, 1997 (unpublished)] is reproduced qualitatively by our reaction temperatures.

The pore of the leaf cavity of Azolla species: teat cell differentiation and cell wall projections.

The differentiation of the specialized secretory teat cells of the leaf cavity pore of Azolla species was investigated at the ultrastructural level with emphasis on their peculiar cell wall projections. The results indicated that the projections are formed as soon as the teat cells complete their differentiation and that their production is principally associated with changes in endoplasmic reticulum profiles. The number of projections increases with the teat cell age and is stimulated under salt and P deficiency stresses. Salt stress also promotes their emergence on Azolla species that under normal conditions do not produce projections. Cytochemical tests on different Azolla species showed that the projection composition is almost identical: proteins, acidic polysaccharides, and pectin are always detected. This study revealed that Azolla teat cell projections differ fundamentally from other types of hitherto described cell wall projections that are considered as remnant structures from cell separation. In contrast, in Azolla teat cells projections are actively produced and compounds are excreted by an exocytotic mechanism. The possible role of the projections in the symbiosis of Azolla spp. with Anabaena azollae is discussed.

Effect of alpha-, beta- and gamma-cyclodextrins on oxygen evolution by the thylakoid membrane. influence of pH and temperature.

The present work investigates the effect of alpha-, beta- and gamma-cyclodextrins (CD), i.e., alpha-CD, beta-CD and gamma-CD, on the oxygen evolution activity, the protein content and the uv-vis spectroscopic characteristics of thylakoid membranes. To study the pH-dependence, the thylakoids were incubated with the cyclodextrins at 273 K for a period of 10 min in the pH range from 5.5 to 9.0. To study the temperature-dependence the membranes were incubated at 273 and 293 K at pH 6.5, that is, the pH which induces a maximal oxygen evolution in the thylakoid preparations. The major observations are: (i) a stimulation of oxygen evolution in thylakoids incubated with alpha- and beta-CD either in acidic or alkaline conditions, (ii) a low inhibitory effect induced by gamma-CD on oxygen evolution, (iii) a significant decrease of the stimulatory effect of alpha- and beta-CD on oxygen evolution as the incubation temperature is raised from 273 to 293 K, (iv) the apparent inability of the cyclodextrins to change the protein contents of the thylakoids, and (v) a significant CD-induced red-shift from 681 to 683 nm observed in the absorption and second derivative spectra of the thylakoid membranes treated with beta-CD. First, it was found that the temperature effect described here is in accord with the general trend of the chemical effect of various cyclodextrins, i.e., the increase of the CD efficiency with decreasing temperature. Secondly, the CD effect is related to the size of the inner cavity diameter of the cyclodextrin molecules. An important conclusion in this work is that the molecular targets of the cyclodextrins are not limited to the thylakoid lipids as was described previously [Rawyler A. and Siegenthaler PA. (1996) Biochim. Biophys. Acta 1278, 89-97], but are located as well in other molecular species exposed at the stromal side of the thylakoid membrane. In particular, the CD-induced red-shift from 681 to 683 nm in the absorption and second derivative spectra of the thylakoid membranes indicates that the cyclodextrins targets might be either the exposed heme macrocycle in cytochrome b559, or the chlorophylls and pheophytins in the pigment-proteins of the photosystems I and II.

Nanoerythrosomes, a new derivative of erythrocyte ghost: IV. Fate of reinjected nanoerythrosomes.

Recently, we have developed a promising new drug carrier named nanoerythrosome (nEryt). This transporter are small vesicles made with the red blood cell membrane. Anticancer drugs like daunorubicin, linked to these nEryt, have a higher antineoplastic activity than the free drug. In this paper, we first analyzed the biodistribution of 125I-nEryt purified by dialysis following intravenous (i.v.) or intraperitoneal (i.p.) injections in CD1 mice. After i.v. administration, nEryt, are rapidly removed from blood circulation (< 30 min). Mainly the liver and spleen take up the vesicles. I.p. injections of nEryt purified by dialysis, showed a marked activity in the inguinal lymph nodes 2 hours post-injection. nEryt purified by centrifugation have a different biodistribution. They accumulate also in the lungs. We demonstrated that accumulation in the lungs is due to particle aggregation during the preparation procedure. Comparative analysis of size distribution of each nEryt preparation revealed that nEtyt purified by centrifugation has a mean diameter of 1.5 microm which is 10 times higher than its dialyzed counterpart. Light microscopic autoradiographs of dialyzed nEryt, reinjected i.v., showed accumulation of nEryt in the sinusoidal lumen as well as in the parenchymal cells of the liver. Autoradiographs of the spleen revealed that nEryt are distributed specifically near the marginal zone and that some of them have escaped the meshes of the red pulp cords.

Quantitative analysis of the stabilization by substrate of Staphylococcus aureus PC1 beta-lactamase.

BACKGROUND: The stabilization of enzymes in the presence of substrates has been recognized for a long time. Quantitative information regarding this phenomenon is, however, rather scarce since the enzyme destroys the potential stabilizing agent during the course of the experiments. In this work, enzyme unfolding was followed by monitoring the progressive decrease of the rate of substrate utilization by the Staphylococcus aureus PC1 beta-lactamase, at temperatures above the melting point of the enzyme. RESULTS: Enzyme inactivation was directly followed by spectrophotometric measurements. In the presence of substrate concentrations above the K(m) values, significant stabilization was observed with all tested compounds. A combination of unfolding kinetic measurements and enzymatic studies, both under steady-state and non-steady-state regimes, allowed most of the parameters characteristic of the two concurrent phenomena (i.e. substrate hydrolysis and enzyme denaturation) to be evaluated. In addition, molecular modelling studies show a good correlation between the extent of stabilization, and the magnitude of the energies of interaction with the enzyme. CONCLUSIONS: Our analysis indicates that the enzyme is substantially stabilized towards heat-induced denaturation, independently of the relative proportions of non-covalent Henri-Michaelis complex (ES) and acyl-enzyme adduct (ES*). Thus, for those substrates with which the two catalytic intermediates are expected to be significantly populated, both species (ES and ES*) appear to be similarly stabilized. This analysis contributes a new quantitative approach to the problem.