Chromosomal genotoxicity of nitrobenzene and benzonitrile

In order to investigate the chromosomal genotoxicity of nitrobenzene and benzonitrile, we studied the induction of micronuclei (MN) by these test compounds in V79 cells, as well as effects on the formation and stability of microtubules and on motor protein functions. No cytotoxicity was seen in V79 cell cultures in terms of Neutral red uptake after 18 h treatment with up to 1 mM nitrobenzene or 1 mM benzonitrile. Subsequently, a concentration range up to 100 muM was used in the experiments on induction of MN. Both test compounds exhibit a weak, but definitely positive test result compared to the solvent (DMSO) control. Minimal effect concentrations of nitrobenzene and benzonitrile appeared as low as 0.01 muM, and no-effect-concentrations were between 0.001 and 0.005 muM. Clearly enhanced MN rates were found at 0.1 muM and higher. Both, nitrobenzene and benzonitrile, induced mostly kinetochor (CREST)-positive micronuclei, thus characterising the chromosomal effects as aneugenic. In cell-free assays, a slight effect on tubulin assembly was observed at 1 mM nitrobenzene without addition of DMSO. Higher concentrations (5 mM) led to secondary effects. In presence of 1% DMSO, nitrobenzene exerted no detectable effect on tubulin assembly up to the solubility limit in water of about 15 mM. For benzonitrile in presence of DMSO, a clear dose-response of inhibition of tubulin assembly at 37degreesC was seen above the no-effect-concentration of 2 mM, with an IC50 of 13 mM and protein denaturation starting above a level of about 20 mM. The nature of the effects of nitrobenzene and benzonitrile on the association of tubulin to form microtubules was confirmed by electron microscopy. Treatment by either 5 mM nitrobenzene or 13 mM benzonitrile plus 1% DMSO left the microtubular structure intact whereas 5 mM nitrobenzene, in absence of DMSO, led to irregular cluster formations. The experiments demonstrate that both nitrobenzene and benzonitrile, in millimolar concentration ranges, may lead to interference with tubulin assembly in a cell-free system. The functionality of the tubulin-kinesin motor protein system was assessed using the microtubule gliding assay. Nitrobenzene affected the gliding velocity in a concentration-dependent manner, starting at about 7.5 muM and reaching complete inhibition of motility at 30 muM, whereas benzonitrile up to 200 muM did not affect the kinesin-driven gliding velocity. The micronucleus assay data demonstrate a chromosomal endpoint of genotoxicity of nitrobenzene and benzonitrile. Aneugenic effects of both compounds occur at remarkably low concentrations, with lowest-effect-concentrations being 0.1 muM. This points to the relevance of interactions with the cellular spindle apparatus.

Disturbed microtubule function and induction of micronuclei by chelate complexes of mercury(II)

Interactions of mercury(II) with the microtubule network of cells may lead to genotoxicity. Complexation of mercury(II) with EDTA is currently being discussed for its employment in detoxification processes of polluted sites. This prompted us to re-evaluate the effects of such complexing agents on certain aspects of mercury toxicity, by examining the influences of mercury(H) complexes on tubulin assembly and kinesin-driven motility of microtubules. The genotoxic effects were studied using the micronucleus assay in V79 Chinese hamster fibroblasts. Mercury(II) complexes with EDTA and related chelators interfered dose-dependently with tubulin assembly and microtubule motility in vitro. The no-effect-concentration for assembly inhibition was 1muM of complexed Hg(II), and for inhibition of motility it was 0.05 muM, respectively. These findings are supported on the genotoxicity level by the results of the micronucleus assay, with micronuclei being induced dose-dependently starting at concentrations of about 0.05 muM of complexed Hg(II). Generally, the no-effect-concentrations for complexed mercury(II) found in the cell-free systems and in cellular assays (including the micronucleus test) were identical with or similar to results for mercury tested in the absence of chelators. This indicates that mercury(II) has a much higher affinity to sulfhydryls of cytoskeletal proteins than to this type of complexing agents. Therefore, the suitability of EDTA and related compounds for remediation of environmental mercury contamination or for other detoxification purposes involving mercury has to be questioned. (C) 2004 Elsevier B.V. All rights reserved.

Genotoxicity of inorganic mercury salts based on disturbed microtubule function

This study investigated the hypothesis that the chromosomal genotoxicity of inorganic mercury results from interaction(s) with cytoskeletal proteins. Effects of Hg2+ salts on functional activities of tubulin and kinesin were investigated by determining tubulin assembly and kinesin-driven motility in cell-free systems. Hg2+ inhibits microtubule assembly at concentrations above 1 muM, and inhibition is complete at about 10 muM. In this range, the tubulin assembly is fully ( up to 6 muM) or partially (similar to 6 - 10 muM) reversible. The inhibition of tubulin assembly by mercury is independent of the anion, chloride or nitrate. The no-observed-effect-concentration for inhibition of microtubule assembly in vitro was 1 muM Hg2+, the IC50 5.8 muM. Mercury(II) salts at the IC50 concentrations partly inhibiting tubulin assembly did not cause the formation of aberrant microtubule structures. Effects of mercury salts on the functionality of the microtubule motility apparatus were studied with the motor protein kinesin. By using a gliding assay'' mimicking intracellular movement and transport processes in vitro, HgCl2 affected the gliding velocity of paclitaxel-stabilised microtubules in a clear dose-dependent manner. An apparent effect is detected at a concentration of 0.1 muM and a complete inhibition is reached at 1 muM. Cytotoxicity of mercury chloride was studied in V79 cells using neutral red uptake, showing an influence above 17 muM HgCl2. Between 15 and 20 muM HgCl2 there was a steep increase in cell toxicity. Both mercury chloride and mercury nitrate induced micronuclei concentration-dependently, starting at concentrations above 0.01 muM. CREST analyses on micronuclei formation in V79 cells demonstrated both clastogenic (CREST-negative) and aneugenic effects of Hg2+, with some preponderance of aneugenicity. A morphological effect of high Hg2+ concentrations ( 100 muM HgCl2) on the microtubule cytoskeleton was verified in V79 cells by immuno-fluorescence staining. The overall data are consistent with the concept that the chromosomal genotoxicity could be due to interaction of Hg2+ with the motor protein kinesin mediating cellular transport processes. Interactions of Hg2+ with the tubulin shown by in vitro investigations could also partly influence intracellular microtubule functions leading, together with the effects on the kinesin, to an impaired chromosome distribution as shown by the micronucleus test.

Genotoxicity of inorganic lead salts and disturbance of microtubule function

Lead compounds are known genotoxicants, principally affecting the integrity of chromosomes. Lead chloride and lead acetate induced concentration-dependent increases in micronucleus frequency in V79 cells, starting at 1.1 μ M lead chloride and 0.05 μ M lead acetate. The difference between the lead salts, which was expected based on their relative abilities to form complex acetato-cations, was confirmed in an independent experiment. CREST analyses of the micronuclei verified that lead chloride and acetate were predominantly aneugenic (CREST-positive response), which was consistent with the morphology of the micronuclei (larger micronuclei, compared with micronuclei induced by a clastogenic mechanism). The effects of high concentrations of lead salts on the microtubule network of V79 cells were also examined using immunofluorescence staining. The dose effects of these responses were consistent with the cytotoxicity of lead(II), as visualized in the neutral-red uptake assay. In a cell-free system, 20-60 μ M lead salts inhibited tubulin assembly dose-dependently. The no-observed-effect concentration of lead(II) in this assay was 10 μ M. This inhibitory effect was interpreted as a shift of the assembly/disassembly steady-state toward disassembly, e.g., by reducing the concentration of assembly-competent tubulin dimers. The effects of lead salts on microtubule-associated motor-protein functions were studied using a kinesin-gliding assay that mimics intracellular transport processes in vitro by quantifying the movement of paclitaxel-stabilized microtubules across a kinesin-coated glass surface. There was a dose-dependent effect of lead nitrate on microtubule motility. Lead nitrate affected the gliding velocities of microtubules starting at concentrations above 10 μ M and reached half-maximal inhibition of motility at about 50 μ M. The processes reported here point to relevant interactions of lead with tubulin and kinesin at low dose levels. Environ. Mal. Mutagen. 45:346-353, 2005. © 2005 Wiley-Liss, Inc.

Evaluation of immune effects of fowlpox vaccine strains and field isolates

The immune effects of fowlpox virus (FPV) field isolates and vaccine strains were evaluated in chickens infected at the age of 1 day and 6 weeks. The field isolates and the obsolete vaccine strain (FPV S) contained integrated reticuloendotheliosis virus (REV) provirus, while the current vaccine strain (FPVST) carries only REV LTR sequences. An indirect antibody ELISA was used to measure the FPV-specific antibody response. The non-specific humoral response was evaluated by injection of two T-cell-dependent antigens, sheep red blood cells (SRBC) and bovine serum albumin (BSA). There was no significant difference in the antibody response to FPV between chickens infected with FPV various isolates and strains at either age. In contrast, antibody responses to both SRBC and BSA were significantly lower in 1-day-old chickens inoculated with field isolates and FPV S at 2-3 weeks post-inoculation. Furthermore, cell-mediated immune (CMI) responses measured by in vitro lymphocyte proliferation assay and in vivo using a PHA-P skin test were significantly depressed in chickens inoculated with field isolates and FPV S at the same periods. In addition, thymus and bursal weights were lower in infected chickens. These immunosuppressive effects were not observed in chickens inoculated with the current vaccine strain, FPVST, at any time. The results of this study suggest that virulent field isolates and FPV S have immunosuppressive effects when inoculated into young chickens, which appeared in the first 3 weeks post infection. REV integrated in the FPV field isolates and FPV S may have played a central role in the development of immunosuppression. (c) 2006 Elsevier B.V. All rights reserved.

Effects of glutathione, N-acetyl-cysteine, α-lipoic acid and dihydrolipoic acid on the cytotoxicity of a 2-pyridylcarboxamidrazone antimycobacterial agent in human mononuclear leucocytes in vitro

A series of antioxidants was used to explore the cytotoxicity of one particularly toxic antimycobacterial 2-pyridylcarboxamidrazone anti-tuberculosis agent against human mononuclear leucocytes (MNL), in comparison with isoniazid (INH) to aid future compound design. INH caused a significant reduction of nearly 40% in cell recovery compared with control (P < 0.0001), although the co-incubation with either glutathione (GSH, 1 mM) or (NAC, 1 mM) showed abolition of INH toxicity. In contrast, the addition of GSH or NAC 1 h after INH failed to protect the cells from INH toxicity (P < 0.0001). The 2-pyridyl-carboxamidrazone 'Compound 1' caused a 50% reduction in cell recovery compared with control (P < 0.001), although this was abolished by the presence of either GSH or NAC. A 1 h post incubation with either NAC or GSH after Compound 1 addition failed to protect the cells from toxicity (P < 0.001). Co-administration of lipoic acid (LA) abolished Compound 1-mediated toxicity, although again, this effect did not occur after LA addition 1 h post incubation with Compound 1 (P < 0.001). However, co-administration of dihydrolipoic acid (DHLA) prevented Compound 1-mediated cell death when incubated with the compound and also after 1 h of Compound 1 alone. Pre-treatment with GSH, then removal of the antioxidant resulted in abolition of Compound 1 toxicity (vehicle control, 63.6 ± 16.7 versus Compound 1 alone 26.1 ± 13.6% versus GSH pre-treatment, 65.7 ± 7.3%). In a cell-free incubation, NMR analysis revealed that GSH does not react with Compound 1, indicating that this agent is not likely to directly deplete membrane thiols. Compound 1's MNL toxicity is more likely to be linked with changes in cell membrane conformation, which may induce consequent thiol depletion that is reversible by exogenous thiols. © 2004 Elsevier B.V. All rights reserved.

Enterotoxigenic Escherichia coli infection of pigs: expression, extraction, purification and the adhesive properties of the K88 fimbrial adhesin

The ability of Escherichia coli to express the K88 fimbrial adhesin was satisfactorily indicated by the combined techniques of ELISA, haemagglutination and latex agglutination. Detection of expression by electron microscopy and the ability to metabolize raffinose were unsuitable. Quantitative expression of the K88 adhesin was determined by ELISA. Expression was found to vary according to the E.coli strain examined, media type and form. In general it was found that the total amount was greater, while the amount/cfu was less on agar than in broth cultures. Expression of the K88 adhesin during unshaken batch culture was related to the growth rate and was maximal during late logarithmic to early stationary phase. A combination of heat extraction, ammonium sulphate and isoelectric precipitation was found suitable for both large and small scale preparation of purified K88ab adhesin. Extraction of the K88 adhesin was sensitive to pH and it was postulated that this may affect the site of colonisation of by ETEC in vivo. Results of haemagglutination experiments were consistent with the hypothesis that the K88 receptor present on erythrocytes is composed of two elements, one responsible for the binding of K88ab and K88ac and a second responsible for the binding of the K88ad adhesin. Comparison of the haemagglutinating properties of cell-free and cell-bound K88 adhesin revealed some differences probably indicating a minor conformational change in the K88 adhesin on its isolation. The K88ab adhesin was found to bind to erythrocytes over a wide pH range (PH 4-9) and was inhibited by αK88ab and αK88b antisera. Inhibition of haemagglutination was noted with crude heparin, mannan and porcine gastric mucin, chondrosine and several hexosamines, glucosamine in particular. The most potent inhibitor of haemagglutination was n-dodecyl-β-D-glucopyranoside, one of a series of glucosides found to have inhibitory properties. Correlation between hydrophobicity of glucosides tested and degree of inhibition observed suggested hydrophobic forces were important in the interaction of the K88 adhesin with its receptor. The results of Scatchard and Hill plots indicated that binding of the K88ab adhesin to porcine enterocytes in the majority of cases is a two-step, three component system. The first K88 receptor (or site) had a K2. of 1.59x1014M-1 and a minimum of 4.3x104 sites/enterocyte. The second receptor (or site) had a K2 of 4.2x1012M-1 with a calculated 1.75x105 sites/enterocyte. Attempts to inhibit binding of cell-free K88 adhesin to porcine enterocytes by lectins were unsuccessful. However, several carbohydrates including trehalose, lactulose, galactose 1→4 mannopyranoside, chondrosine, galactosamine, stachyose and mannan were inhibitory. The most potent inhibitor was found to be porcine gastric mucin. Inhibition observed with n-octyl-α-D-glucopyranose was difficult to interpret in isolation because of interference with the assay, however, it agreed with the results of haemagglutination inhibition experiments.

Dehdyroepiandrosterone sulfate directly activates protein kinase C-β to increase human neutrophil superoxide generation

Dehydroepiandrosterone sulfate (DHEAS) is the most abundant steroid in the human circulation and is secreted by the adrenals in an age-dependent fashion, with maximum levels during the third decade and very low levels in old age. DHEAS is considered an inactive metabolite, whereas cleavage of the sulfate group generates dehydroepiandrosterone (DHEA), a crucial sex steroid precursor. However, here we show that DHEAS, but not DHEA, increases superoxide generation in primed human neutrophils in a dose-dependent fashion, thereby impacting on a key bactericidal mechanism. This effect was not prevented by coincubation with androgen and estrogen receptor antagonists but was reversed by the protein kinase C inhibitor Bisindolylmaleimide 1. Moreover, we found that neutrophils are unique among leukocytes in expressing an organic anion-transporting polypeptide D, able to mediate active DHEAS influx transport whereas they did not express steroid sulfatase that activates DHEAS to DHEA. A specific receptor for DHEAS has not yet been identified, but we show that DHEAS directly activated recombinant protein kinase C-ß (PKC-ß) in a cell-free assay. Enhanced PKC-ß activation by DHEAS resulted in increased phosphorylation of p47phox, a crucial component of the active reduced nicotinamide adenine dinucleotide phosphate complex responsible for neutrophil superoxide generation. Our results demonstrate that PKC-ß acts as an intracellular receptor for DHEAS in human neutrophils, a signaling mechanism entirely distinct from the role of DHEA as sex steroid precursor and with important implications for immunesenescence, which includes reduced neutrophil superoxide generation in response to pathogens.

The integration of ProxiMAX randomisation with CIS display for the production of novel peptides

Saturation mutagenesis is a powerful tool in modern protein engineering, which permits key residues within a protein to be targeted in order to potentially enhance specific functionalities. However, the creation of large libraries using conventional saturation mutagenesis with degenerate codons (NNN or NNK/S) has inherent redundancy and consequent disparities in codon representation. Therefore, both chemical (trinucleotide phosphoramidites) and biological methods (sequential, enzymatic single codon additions) of non-degenerate saturation mutagenesis have been developed in order to combat these issues and so improve library quality. Large libraries with multiple saturated positions can be limited by the method used to screen them. Although the traditional screening method of choice, cell-dependent methods, such as phage display, are limited by the need for transformation. A number of cell-free screening methods, such as CIS display, which link the screened phenotype with the encoded genotype, have the capability of screening libraries with up to 1014 members. This thesis describes the further development of ProxiMAX technology to reduce library codon bias and its integration with CIS display to screen the resulting library. Synthetic MAX oligonucleotides are ligated to an acceptor base sequence, amplified, and digested, subsequently adding a randomised codon to the acceptor, which forms an iterative cycle using the digested product of the previous cycle as the base sequence for the next. Initial use of ProxiMAX highlighted areas of the process where changes could be implemented in order to improve the codon representation in the final library. The refined process was used to construct a monomeric anti-NGF peptide library, based on two proprietary dimeric peptides (Isogenica) that bind NGF. The resulting library showed greatly improved codon representation that equated to a theoretical diversity of ~69%. The library was subsequently screened using CIS display and the discovered peptides assessed for NGF-TrkA inhibition by ELISA. Despite binding to TrkA, these peptides showed lower levels of inhibition of the NGF-TrkA interaction than the parental dimeric peptides, highlighting the importance of dimerization for inhibition of NGF-TrkA binding.

Optimisation of a droplet digital PCR assay for the diagnosis of Schistosoma japonicum infection: A duplex approach with DNA binding dye chemistry

Schistosomiasis is a chronically debilitating helminth infection with a significant socio-economic and public health impact. Accurate diagnostics play a pivotal role in achieving current schistosomiasis control and elimination goals. However, many of the current diagnostic procedures, which rely on detection of schistosome eggs, have major limitations including lack of accuracy and the inability to detect pre-patent infections. DNA-based detection methods provide a viable alternative to the current tests commonly used for schistosomiasis diagnosis. Here we describe the optimisation of a novel droplet digital PCR (ddPCR) duplex assay for the diagnosis of Schistosoma japonicum infection which provides improved detection sensitivity and specificity. The assay involves the amplification of two specific and abundant target gene sequences in S. japonicum; a retrotransposon (SjR2) and a portion of a mitochondrial gene (nad1). The assay detected target sequences in different sources of schistosome DNA isolated from adult worms, schistosomules and eggs, and exhibits a high level of specificity, thereby representing an ideal tool for the detection of low levels of parasite DNA in different clinical samples including parasite cell free DNA in the host circulation and other bodily fluids. Moreover, being quantitative, the assay can be used to determine parasite infection intensity and, could provide an important tool for the detection of low intensity infections in low prevalence schistosomiasis-endemic areas.

Circulating miRNAs: Potential Novel Biomarkers for Hepatopathology Progression and Diagnosis of Schistosomiasis Japonica in Two Murine Models

BACKGROUND: Schistosomiasis remains a major public health issue, with an estimated 230 million people infected worldwide. Novel tools for early diagnosis and surveillance of schistosomiasis are currently needed. Elevated levels of circulating microRNAs (miRNAs) are commonly associated with the initiation and progression of human disease pathology. Hence, serum miRNAs are emerging as promising biomarkers for the diagnosis of a variety of human diseases. This study investigated circulating host miRNAs commonly associated with liver diseases and schistosome parasite-derived miRNAs during the progression of hepatic schistosomiasis japonica in two murine models.

METHODOLOGY/PRINCIPAL FINDINGS: Two mouse strains (C57BL/6 and BALB/c) were infected with a low dosage of Schistosoma japonicum cercariae. The dynamic patterns of hepatopathology, the serum levels of liver injury-related enzymes and the serum circulating miRNAs (both host and parasite-derived) levels were then assessed in the progression of schistosomiasis japonica. For the first time, an inverse correlation between the severity of hepatocyte necrosis and the level of liver fibrosis was revealed during S. japonicum infection in BALB/c, but not in C57BL/6 mice. The inconsistent levels of the host circulating miRNAs, miR-122, miR-21 and miR-34a in serum were confirmed in the two murine models during infection, which limits their potential value as individual diagnostic biomarkers for schistosomiasis. However, their serum levels in combination may serve as a novel biomarker to mirror the hepatic immune responses induced in the mammalian host during schistosome infection and the degree of hepatopathology. Further, two circulating parasite-specific miRNAs, sja-miR-277 and sja-miR-3479-3p, were shown to have potential as diagnostic markers for schistosomiasis japonica.

CONCLUSIONS/SIGNIFICANCE: We provide the first evidence for the potential of utilizing circulating host miRNAs to indicate different immune responses and the severity of hepatopathology outcomes induced in two murine strains infected with S. japonicum. This study also establishes a basis for the early and cell-free diagnosis of schistosomiasis by targeting circulating schistosome parasite-derived miRNAs.

Avaliação do padrão de resposta de imunoglobulinas em diferentes linhagens de camundongos frente à infecção por T.cruzi

Dissertação (Mestrado)

Computer simulations of soft particles in flow

Understanding the dynamics of blood cells is a crucial element to discover biological mechanisms, to develop new efficient drugs, design sophisticated microfluidic devices, for diagnostics. In this work, we focus on the dynamics of red blood cells in microvascular flow. Microvascular blood flow resistance has a strong impact on cardiovascular function and tissue perfusion. The flow resistance in microcirculation is governed by flow behavior of blood through a complex network of vessels, where the distribution of red blood cells across vessel cross-sections may be significantly distorted at vessel bifurcations and junctions. We investigate the development of blood flow and its resistance starting from a dispersed configuration of red blood cells in simulations for different hematocrits, flow rates, vessel diameters, and aggregation interactions between red blood cells. Initially dispersed red blood cells migrate toward the vessel center leading to the formation of a cell-free layer near the wall and to a decrease of the flow resistance. The development of cell-free layer appears to be nearly universal when scaled with a characteristic shear rate of the flow, which allows an estimation of the length of a vessel required for full flow development, $l_c \approx 25D$, with vessel diameter $D$. Thus, the potential effect of red blood cell dispersion at vessel bifurcations and junctions on the flow resistance may be significant in vessels which are shorter or comparable to the length $l_c$. The presence of aggregation interactions between red blood cells lead in general to a reduction of blood flow resistance. The development of the cell-free layer thickness looks similar for both cases with and without aggregation interactions. Although, attractive interactions result in a larger cell-free layer plateau values. However, because the aggregation forces are short-ranged at high enough shear rates ($\bar{\dot{\gamma}} \gtrsim 50~\text{s}^{-1}$) aggregation of red blood cells does not bring a significant change to the blood flow properties. Also, we develop a simple theoretical model which is able to describe the converged cell-free-layer thickness with respect to flow rate assuming steady-state flow. The model is based on the balance between a lift force on red blood cells due to cell-wall hydrodynamic interactions and shear-induced effective pressure due to cell-cell interactions in flow. We expect that these results can also be used to better understand the flow behavior of other suspensions of deformable particles such as vesicles, capsules, and cells. Finally, we investigate segregation phenomena in blood as a two-component suspension under Poiseuille flow, consisting of red blood cells and target cells. The spatial distribution of particles in blood flow is very important. For example, in case of nanoparticle drug delivery, the particles need to come closer to microvessel walls, in order to adhere and bring the drug to a target position within the microvasculature. Here we consider that segregation can be described as a competition between shear-induced diffusion and the lift force that pushes every soft particle in a flow away from the wall. In order to investigate the segregation, on one hand, we have 2D DPD simulations of red blood cells and target cell of different sizes, on the other hand the Fokker-Planck equation for steady state. For the equation we measure force profile, particle distribution and diffusion constant across the channel. We compare simulation results with those from the Fokker-Planck equation and find a very good correspondence between the two approaches. Moreover, we investigate the diffusion behavior of target particles for different hematocrit values and shear rates. Our simulation results indicate that diffusion constant increases with increasing hematocrit and depends linearly on shear rate. The third part of the study describes development of a simulation model of complex vascular geometries. The development of the model is important to reproduce vascular systems of small pieces of tissues which might be gotten from MRI or microscope images. The simulation model of the complex vascular systems might be divided into three parts: modeling the geometry, developing in- and outflow boundary conditions, and simulation domain decomposition for an efficient computation. We have found that for the in- and outflow boundary conditions it is better to use the SDPD fluid than DPD one because of the density fluctuations along the channel of the latter. During the flow in a straight channel, it is difficult to control the density of the DPD fluid. However, the SDPD fluid has not that shortcoming even in more complex channels with many branches and in- and outflows because the force acting on particles is calculated also depending on the local density of the fluid.

Messenger ribonucleic acid of cerebral nuclei.

1. RNA was isolated from crude nuclear preparations and from ribosomes derived from rat brain and liver. Nuclear RNA was obtained by lysis of the nuclei with sodium dodecyl sulphate, followed by denaturation and removal of DNA and protein with hot phenol. 2. Base composition analyses indicated that the cerebral nuclear RNA preparation contained a higher proportion of non-ribosomal RNA than the analogous hepatic preparation. 3. Sucrose-density-gradient analyses revealed a heterogeneous profile for each nuclear RNA preparation, with two major peaks possessing the sedimentation properties of ribosomal RNA (18s and 28s). 4. Template activities of both preparations were widely distributed through the sucrose density gradients. 5. The cerebral nuclear RNA preparation was more active than the hepatic nuclear RNA preparation in promoting amino acid incorporation in cell-free systems from Escherichia coli and rat brain. 6. Cerebral nuclear RNA stimulated amino acid incorporation in a cerebral ribosomal system even in the presence of an excess of purified E. coli transfer RNA. 7. It is concluded that a significant proportion of cerebral nuclear RNA has the characteristics of messenger RNA.

Engineered Vesicles for Perchlorate Degradation

This study demonstrates the use of engineered vesicles to reduce perchlorate. Specifically, cell-free extracts containing perchlorate reductase and chlorite dismutase enzymes were encapsulated in a triblock copolymer vesicle functionalized with the outer membrane porin OmpF. The porin allows for perchlorate transport into the vesicles, inside which the encapsulated enzymes transform perchlorate to chloride. Perchlorate reduction was quantified using a methyl viologen colorimetric technique. The vesicle solutions had perchlorate-reducing activities ranging from 35-45 units per liter. This work shows that vesicles can provide a mechanism to utilize environmentally-relevant biological enzymes. When incorporated into a vesicle, the enzymes could be used outside of environmental conditions where they would normally be expressed by natural bacteria.