Enhanced downregulation of the p75 nerve growth factor receptor by cholesteryl and bis-cholesteryl antisense oligonucleotides

The effects of conjugating cholesterol to either or both ends of a phosphorothioate (PS) oligonucleotide were analyzed in terms of cellular uptake and antisense efficacy. The oligo sequence was directed against the p75 nerve growth factor receptor (p75), and was tested in differentiated PC12 cells, which express high levels of this protein. The addition of a single cholesteryl group to the 5'-end significantly increased cellular uptake and improved p75 mRNA downregulation compared with the unmodified PS oligo, However, only a minor degree of downregulation of p75 protein was obtained with 5' cholesteryl oligos, Three different linkers was used to attach the 5' cholesteryl group but were found not to have any impact on efficacy. Addition of a single cholesteryl group to the 3'-end led to greater p75 mRNA downregulation (31%) and p75 protein downregulation (28%) than occurred with the 5' cholesteryl oligos. The biggest improvement in antisense efficacy, both at the mRNA and protein levels, was obtained from the conjugation of cholesterol to both ends of the oligo. One of the bis-cholesteryl oligos was nearly as effective as cycloheximide at decreasing synthesis of p75, The bis-cholesteryl oligos also displayed significant efficacy at 1 mu M, whereas the other oligos required 5 mu M to be effective. The enhanced efficacy of bis-cholesteryl oligos is likely to be due to a combination of enhanced cellular uptake and resistance to both 5' and 3' exonucleases.

Experimental study of phase equilibria in the system PbO-ZnO-SiO2

An experimental study on the ternary system PbO-ZnO-SiO2, in air by high-temperature equilibration and quenching techniques followed by electron probe X-ray microanalysis was carried out as part of the wider research program on the six-component system PbO-ZnO-SiO2-CaO-FeO-Fe2O3, which combines experimental and thermodynamic computer modeling techniques to characterize zinc and lead industrial slags. Liquidus and solidus data were reported for all primary phase fields in the system PbO-ZnO-SiO2 in the temperature range 640 degrees C to 1400 degrees C (913 to 1673 K).

The crack tip strain field of AISI 4340 - Part II - Experimental measurements

Crack tip strain maps have been measured for AISI 4340 high strength steel. No significant creep was observed. The measured values of CTOD were greater than expected from the HRR model. Crack tip branching was observed in every experiment. The direction of crack branching was in the same direction as a major ridge'' of epsilon(yy) strain, which in turn was in the same direction as predicted by the HRR model. Furthermore, the measured magnitudes of the epsilon(y)y strain in this same direction were in general greater than the values predicted by the HRR model. This indicates more plasticity in the crack tip region than expected from the HRR model. This greater plasticity could be related to the larger than expected CTOD values. The following discrepancies between the measured strain fields for AISI 4340 and the HRR predictions are noteworthy: (1) The crack branching. (2) Values of CTOD significantly higher than predicted by HRR. (3) The major ridge'' of epsilon(yy) strain an angle of about 60 degrees with the direction of overall propagation of the fatigue precrack, in which the measured magnitudes of the epsilon(yy) strain were greater than the values predicted by the HRR model. (4) Asymmetric shape of the plastic zone as measured by the epsilon(yy) strain. (5) Values of shear strain gamma(xy) significantly higher than predicted by the HRR model. (C) 1999 Kluwer Academic Publishers.

Numerical and experimental study of seepage in unconfined aquifers with a periodic boundary condition

The assessment of groundwater conditions within an unconfined aquifer with a periodic boundary condition is of interest in many hydrological and environmental problems. A two-dimensional numerical model for density dependent variably saturated groundwater flow, SUTRA (Voss, C.I., 1984. SUTRA: a finite element simulation model for saturated-unsaturated, fluid-density dependent ground-water flow with energy transport or chemically reactive single species solute transport. US Geological Survey, National Center, Reston, VA) is modified in order to be able to simulate the groundwater flow in unconfined aquifers affected by a periodic boundary condition. The basic flow equation is changed from pressure-form to mixed-form. The model is also adjusted to handle a seepage-face boundary condition. Experiments are conducted to provide data for the groundwater response to the periodic boundary condition for aquifers with both vertical and sloping faces. The performance of the numerical model is assessed using those data. The results of pressure- and mixed-form approximations are compared and the improvement achieved through the mixed-form of the equation is demonstrated. The ability of the numerical model to simulate the water table and seepage-face is tested by modelling some published experimental data. Finally the numerical model is successfully verified against present experimental results to confirm its ability to simulate complex boundary conditions like the periodic head and the seepage-face boundary condition on the sloping face. (C) 1999 Elsevier Science B.V. All rights reserved.

Evolution of the genetic covariance between male and female components of mate recognition: An experimental test

The evolution of a positive genetic correlation between male and female components of mate recognition systems will result as a consequence of assortative mating and, in particular, is central to a number of theories of sexual selection. Although the existence of such genetic correlations has been investigated in a number of taxa, it has yet to be shown that such correlations evolve and whether they may evolve as rapidly as suggested by sexual selection models. In this study, I used a hybridization experiment to disrupt natural mate recognition systems and then observed the subsequent evolutionary dynamics of the genetic correlation between male and female components for 56 generations in hybrids between Drosophila serrata and Drosophila birchii. The genetic correlation between male and female components evolved from 0.388 at generation 5 to 1.017 at generation 37 and then declined to -0.040 after a further 19 generations. These results indicated that the genetic basis of the mate recognition system in the hybrid populations evolved rapidly. The initial rapid increase in the genetic correlation was consistent with the classic assumption that male and female components will coevolve under sexual selection. The subsequent decline in genetic correlation may be attributable to the fixation of major genes or, alternatively, may be a result of a cyclic evolutionary change in mate recognition.

Differential tumor surveillance by natural killer (NK) and NKT cells

Natural tumor surveillance capabilities of the host were investigated in six different mouse tumor models where endogenous interleukin (IL)-12. does or does not dictate the efficiency of the innate immune response. Gene-targeted and lymphocyte subset-depleted mice were used to establish the relative importance of natural killer (NK) and NK1.1(+) T (NKT) cells in protection from tumor initiation and metastasis. In the models examined, CD3(-) NK cells were responsible for tumor rejection and protection from metastasis in models where control of major histocompatibility complex class I-deficient tumors was independent of IL-12, A protective role for NKT cells was only observed when tumor rejection required endogenous IL-12 activity. In particular, T cell receptor J alpha 281 gene-targeted mice confirmed a critical function for NKT cells in protection from spontaneous tumors initiated by the chemical carcinogen, methylcholanthrene. This is the first description of an antitumor function for NKT cells in the absence of exogenously administered potent stimulators such as IL-12 or alpha-galactosylceramide.

Induction of an antigen-specific CTL response by a conformationally biased agonist of human C5a anaphylatoxin as a molecular adjuvant

A conformationally biased decapeptide agonist of human C5a anaphylatoxin (YSPKPMPLaR) was used as a molecular adjuvant in stimulating an Ag-specific CTL response against murine P815S target cells expressing an Ld-restricted CTL epitope of the hepatitis B surface Ag (HBsAg), Groups of BALB/c mice (H-2(d)) were immunized with aqueous solutions of the HBsAg CTL epitopes (IPQSLDSWWTSL and IPQSLDSTaVTSLRR); the C5a agonist (YSFKPMPLaR); the C5a agonist and HBsAg CTL epitopes admired (IPQSLDSWWTSL and IPQSLDSWWTSLRR + YSFKPMPLaR); the C5a-active, HBsAg CTL epitope-C5a agonist constructs (IPQSLDSWWTSLYSFKPMPLaR, IPQSLDSWWTSLRRYSFKPMPLaR, and IPQSLDSWWTSLRVRRYSFPMPLaR); a C5a-inactive, reverse-moiety construct (YSFKPMPLaRRRIPQSLDSWWTSL); and a C5a-attenuated, carboxyl-terminal-blocked construct (IPQSLDSWWTSLRRYSFKPMPLaRG). Ag-specific CD8(+) CTL responses were observed after the secondary boost in the absence of any added adjuvant only in mice that were immunized with C5a-active contructs, IPQSLDSWWTSLRRYSFKPMPLaR and IPQSLDSWWTSLRVRRYSFKPMPLaR. These two C5a-active immunogens contained potential subtilisin-sensitive linker sequences between the HBsAg CTL epitope and the C5a agonist; i.e., a double-Arg (RR) and a furin protease sensitive sequence (RVRR), The introduction of these potentially cleavable sequences may be a method of increasing the likelihood of liberating the CTL epitope from the C5a agonist by intracellular proteases, thereby facilitating entry of the epitope into Ag-processing pathways via an exogenous route.

Sequential increase in Egr-1 and AP-1 DNA binding activity in the dentate gyrus following the induction of long-term potentiation

Establishment of long-term potentiation (LTP) at perforant path synapses is highly correlated with increased expression of Egr and AP-1 transcription factors in rat dentate gyrus granule cells. We have investigated whether increased transcription factor levels are reflected in increased transcription factor activity by assessing Egr and AP-I DNA binding activity using gel shift assays. LTP produced an increase in binding to the Egr element, which was NMDA receptor-dependent and correlated closely with our previously reported increase in Egr-1 (zif/268) protein levels. Supershift analysis confirmed involvement of Egr-1, but not Egr-2 in the DNA binding activity. AP-1 DNA binding was also rapidly elevated in parallel with protein levels, however, the peak increase in activity was delayed until 4 h, a time point when we have previously shown that only jun-D protein was elevated. These data indicate that binding of Egr-1 and AP-1 to their response elements is increased in two phases. This may result in activation of distinct banks of target genes which contribute to the establishment of persistent LTP. (C) 2000 Elsevier Science B.V. All rights reserved.

Experimental examination of solute transport by surface runoff from low-angle slopes

The removal of chemicals in solution, by overland flow from agricultural land has the potential to be a significant source of chemical loss from zero-till and surface mulched farming systems. The objective of this study was to determine the magnitude of solute loss by surface runoff from agricultural systems. Previous experiments have enhanced the understanding of the exchange process, but the initial soil conditions together with the tracer application method in these experiments have meant that in some cases the results have limited applicability to field situations. In this study, two different sets of experiments were carried out to determine the magnitude of solute loss by surface runoff. These experiments entailed the surface application of bromide to (1) field scale plots 18 m long by 2 m wide and (2) repacked soil cores 236 mm in diameter; followed by the application of simulated rainfall in both cases. The most substantial finding of the field experiments was that the quantities of solute in surface runoff varied greatly with soil type and structure (0.07-14.9% of the applied bromide). Also, on some soils, large quantities of tracer were measured in the surface runoff even after several hours of infiltration. The experiments on soil cores showed that soil structure plays an important role in the quantity of chemical that may be transported in the surface runoff. These field results showed that, in certain systems, solute movement by overland flow is an important transport mechanism, which should be considered when budgeting for chemical loss. (C) 2000 Elsevier Science B.V. All rights reserved.

Coupled experimental and thermodynamic modeling studies for metallurgical smelting and coal combustion slag systems

An extensive research program focused on the characterization of various metallurgical complex smelting and coal combustion slags is being undertaken. The research combines both experimental and thermodynamic modeling studies. The approach is illustrated by work on the PbO-ZnO-Al2O3-FeO-Fe2O3-CaO-SiO2 system. Experimental measurements of the liquidus and solidus have been undertaken under oxidizing and reducing conditions using equilibration, quenching, and electron probe X-ray microanalysis. The experimental program has been planned so as to obtain data for thermodynamic model development as well as for pseudo-ternary Liquidus diagrams that can be used directly by process operators. Thermodynamic modeling has been carried out using the computer system FACT, which contains thermodynamic databases with over 5000 compounds and evaluated solution models. The FACT package is used for the calculation of multiphase equilibria in multicomponent systems of industrial interest. A modified quasi-chemical solution model is used for the liquid slag phase. New optimizations have been carried out, which significantly improve the accuracy of the thermodynamic models for lead/zinc smelting and coal combustion processes. Examples of experimentally determined and calculated liquidus diagrams are presented. These examples provide information of direct relevance to various metallurgical smelting and coal combustion processes.

Resident tissue macrophages within the normal rat iris lack immunosuppressive activity and are effective antigen-presenting cells

Despite extensive study of the numerous immunoregulatory mechanisms that contribute to the immune-privileged nature of the anterior chamber (AC) of the eye, little is known of the functional nature of antigen-presenting cells (APC) present in the tissues adjoining the AC. In the present study, we have compared the antigen-presenting capacity of dendritic cells (DC) and macrophages isolated from the normal rat iris. Whereas iris DC exhibited a potent ability to stimulate resting allogeneic T cells in MLR cultures (an in-vitro correlate of the ability to induce primary T cell responses), resident iris macrophages displayed negligible MLR-stimulatory capacity. Significantly, iris macrophages could efficiently elicit proliferation of primed antigen-specific T cells (an in-vitro correlate of the ability to act as local APC in secondary responses). This antigen-presenting activity was approximately half that of fully mature iris DC and considerably greater than that of freshly isolated iris DC. A key contributor to the effectiveness of resident iris macrophage antigen presentation was considered to be the absence of lymphocytostatic control of T cell proliferation exerted by these cells. The results indicate dichotomous but complementary roles for DC (immune surveillance) and macrophages (local antigen presentation in secondary responses) in this tissue.

Experimental study of phase equilibria in the systems Fe-Zn-O and Fe-Zn-Si-O at metallic iron saturation

The reported experimental work on the systems Fe-Zn-O and Fe-Zn-Si-O in equilibrium with metallic iron is part of a wider research program that combines experimental and thermodynamic computer modeling techniques to characterize zinc/lead industrial slags and sinters in the system PbO-ZnO-SiO2-CaO-FeO-Fe2O3. Extensive experimental,investigations using high-temperature equilibration and quenching techniques followed by electron probe X-ray microanalysis (EPMA) were carried out. Special experimental; procedures were developed to enable accurate measurements in these ZnO-containing systems to be performed in equilibrium with metallic iron; The systems Fe-Zn-O and FeZn-Si-O were experimentally investigated in equilibrium with metallic iron in the temperature ranges 900 degreesC to 1200 degreesC (1173 to 1473 K) and from 1000 degreesC to 1350 degreesC (1273 to 1623 K), respectively. The liquidus surface in the system Fe-Zn-Si-O in equilibrium with metallic iron was characterized in the composition ranges 0 to 33 wt pet ZnO and 0 to 40 wt pet SiO2. The wustite (Fe,Zn)O, zincite (Zn,Fe)O, willemite (Zn,Fe)(2)SiO4, arid fayalite: (Fe,Zn)(2)SiO4 solid solutions in equilibrium with metallic iron were measured.

Experimental study of phase equilibria in the PbO-MgO-SiO2 system

Equilibrium phase relations in the PbO-Al2O3-SiO2 system have been investigated experimentally by means of high-temperature equilibration, quenching, and electron probe X-ray microanalysis (EPMA). The system has 21 primary phase fields including three monoxides (PbO, Al2O3, and SiO2), seven binary compounds (Al6Si2O13, PbAl2O4, PbAl12O19, Pb2Al2O5, PbSiO3, Pb2SiO4, and Pb4SiO6), and eleven ternary compounds (PbAl2Si2O8, Pb3Al10SiO20, Pb4Al2Si2O11, Pb4Al4SiO12, Pb4Al4Si3O16, Pb4Al4Si5O20, Pb5Al2Si10O28, Pb6Al2Si6O21, Pb8Al2Si4O19, Pb12Al2Si17O49, and Pb12Al2Si20O55). Three new ternary compounds, Pb4Al4SiO12, Pb4Al4Si5O20, and Pb12Al2Si17O49, were observed and characterized by EPMA. No extensive solid solution in any of the compounds was found in the present study. The liquidus isotherms were experimentally determined in most of the primary phase fields in the temperature range from 923 to 1873 K, and the ternary phase diagram of the PbO-Al2O3-SiO2 System has been constructed.

Experimental methods for studying drug uptake in the head and brain

A number of techniques have been developed to study the disposition of drugs in the head and, in particular, the role of the blood-brain barrier (BBB) in drug uptake. The techniques can be divided into three groups: in-vitro, in-vivo and in-situ. The most suitable method depends on the purpose(s) and requirements of the particular study being conducted. In-vitro techniques involve the isolation of cerebral endothelial cells so that direct investigations of these cells can be carried out. The most recent preparations are able to maintain structural and functional characteristics of the BBB by simultaneously culturing endothelial cells with astrocytic cells,The main advantages of the in-vitro methods are the elimination of anaesthetics and surgery. In-vivo methods consist of a diverse range of techniques and include the traditional Brain Uptake Index and indicator diffusion methods, as well as microdialysis and positron emission tomography. In-vivo methods maintain the cells and vasculature of an organ in their normal physiological states and anatomical position within the animal. However, the shortcomings include renal acid hepatic elimination of solutes as well as the inability to control blood flow. In-situ techniques, including the perfused head, are more technically demanding. However, these models have the ability to vary the composition and flow rate of the artificial perfusate. This review is intended as a guide for selecting the most appropriate method for studying drug uptake in the brain.