A computer-controlled spraying-freezing apparatus for millisecond time-resolution electron cryomicroscopy

The application of gene therapy techniques to the clinical problem of coronary restenosis has generated tremendous attention and enthusiasm. Use of gene transfer technology to prevent a common intractable illness would represent a watershed event for human gene therapy. However, the time is not yet right to initiate gene therapy trials for restenosis. The biology of restenosis is incompletely understood, catheter-based gene delivery is poorly adapted to the coronary circulation, and current gene transfer vectors are ill-suited for safe and effective gene delivery to the coronary artery wall. Basic research designed to overcome these obstacles is currently more appropriate than the initiation of clinical trials.     

Regulation of presynaptic NMDA responses by external and intracellular pH changes at developing neuromuscular synapses

NMDA receptors play important roles in synaptic plasticity and neuronal development. The functions of NMDA receptors are modulated by many endogenous substances, such as external pH (pHe), as well as second messenger systems. In the present study, the nerve-muscle cocultures of Xenopus embryos were used to investigate the effects of both external and intracellular pH (pHi) changes on the functional responses of presynaptic NMDA receptors. Spontaneous synaptic currents (SSCs) were recorded from innervated myocyte using whole-cell recordings. Local perfusion of NMDA at synaptic regions increased the SSC frequency via the activation of presynaptic NMDA receptors. A decrease in pHe from 7.6 to 6.6 reduced NMDA responses to 23% of the control, and an increase in pHe from 7.6 to 8.6 potentiated the NMDA responses in increasing SSC frequency. The effect of NMDA on intracellular Ca2+ concentration ([Ca2+]i) was also affected by pHe changes: external acidification inhibited and alkalinization potentiated [Ca2+]i increases induced by NMDA. Intracellular pH changes of single soma were measured by ratio fluorometric method using 2,7-bis (carboxyethyl)-5, 6-carboxyfluorescein (BCECF). Cytosolic acidification was used in which NaCl in Ringer's solution was replaced with weak organic acids. Acetate and propionate but not methylsulfate substitution caused intracellular acidification and potentiated NMDA responses in increasing SSC frequency, intracellular free Ca2+ concentration, and NMDA-induced currents. On the other hand, cytosolic alkalinization with NH4Cl did not significantly affect these NMDA responses. These results suggest that the functions of NMDA receptors are modulated by both pHe and pHi changes, which may occur in some physiological or pathological conditions.     

Efficient lymphocyte migration across high endothelial venules of mouse Peyer's patches requires overlapping expression of L-selectin and beta7 integrin

Lymphocyte migration into lymphoid organs is regulated by adhesion molecules including L-selectin and the beta7 integrins. L-selectin and alpha4beta7 are predominantly hypothesized to direct the selective migration of lymphocytes to peripheral lymph nodes and the gut-associated lymphoid tissues, respectively. To further characterize interactions between L-selectin and beta7 integrins during lymphocyte recirculation, mice deficient in both receptors (L-selectin/beta7 integrin-/-) were generated. The simultaneous loss of L-selectin and beta7 integrin expression prevented the majority of lymphocytes (>95% inhibition) from attaching to high endothelial venules (HEV) of Peyer's patches and other lymphoid tissues during in vitro binding assays. Moreover, the inability to bind HEV eliminated the vast majority of L-selectin/beta7 integrin-/- lymphocyte migration into Peyer's patches during short-term and long-term in vivo migration assays (>99% inhibition,p < 0.01). The lack of lymphocyte migration into Peyer's patches correlated directly with the dramatically reduced size and cellularity (99% reduced) of this tissue in L-selectin/beta7 integrin-/- mice. High numbers of injected L-selectin/beta7 integrin-/- lymphocytes remaining in the blood of wild-type mice correlated with markedly increased numbers of circulating lymphocytes in L-selectin/beta7 integrin-/- mice. Loss of either L-selectin or the beta7 integrins alone resulted in significant but incomplete inhibition of Peyer's patch migration. Collectively, the phenotype of L-selectin/beta7 integrin-/- mice demonstrates that these two receptors primarily interact along the same adhesion pathway that is required for the vast majority of lymphocyte migration into Peyer's patches.     

Lipopeptide phytotoxins produced by Pseudomonas syringae pv. syringae: comparison of the biosurfactant and ion channel-forming activities of syringopeptin and syringomycin

The maturational status of Adelta and C-fibers in the fetal rat spinal cord was examined using formalin-induced c-fos expression as a marker for neuronal activities. Awake 19-, 20-, and 21-day fetuses (FD) were injected ex utero with 5 microl of 10% formalin either into the ventral aspect of the forepaw or the hindpaw. FD 19 fetuses showed little response to the injection, but with increasing age, the fetuses exhibited more specific behaviors following injury of the paw. By FD 21, fetuses treated with formalin injection showed body curls and twitches, mouth opening, face wiping, and withdrawal of the injected paw. The anatomical data paralleled that of behavior; FD 19 animals expressed a small number of Fos labeled nuclei following the formalin injection that was not statistically different from control animals. The formalin-induced increase in Fos staining was first observed at FD 20 with a large increase in the number of Fos labeled cell occurring between FD 20 and 21. By FD 21, the pattern of Fos stained nuclei resembled that found in neonatal rats. There was constitutive bilateral staining in all untreated, saline and formalin injected fetuses that is unique to prenatal animals. Formalin treated fetuses showed constitutive level of staining in addition to the increase in the c-fos expression caused by formalin. We have thus demonstrated that, as indexed both by behavioral response and by Fos immunoreactivity, rat fetuses are capable of transmitting and responding to noxious input before birth.     

A hermeneutic analysis of the process of conducting clinical interviews

1. Although no right way to interview exists, an interviewer should develop a comfortable, personal style. 2. Allowing an interview to follow its own course while remembering the overall interview agenda will contribute to quality of the data. 3. Interviewing skills are enhanced as the interviewer develops a personal style and becomes comfortable as a practitioner.     

Increased response/recovery lifetimes and reinforcement of polyaniline nanofiber films using carbon nanotubes

We have prepared high surface area, conductive, mechanically robust, responsive polyaniline–carbon nanotube composite films. These were produced by filtration from dilute dispersions of polyaniline nanofibers and single-walled carbon nanotubes. Unlike polyaniline alone, these composites are mechanically stable, maintain large intractable surfaces and exhibit greatly enhanced response/recovery behavior to changes in their local environment. This is illustrated by exposing the films to ammonia.     

Enhancing the carbon sink in European agricultural soils: including trace gas fluxes in estimates of carbon mitigation potential

The possibility that the carbon sink in agricultural soils can be enhanced has taken on great political significance since the Kyoto Protocol was finalised in December 1997. The Kyoto Protocol allows carbon emissions to be offset by demonstrable removal of carbon from the atmosphere. Thus, forestry activities (Article 3.3) and changes in the use of agricultural soils (Article 3.4) that are shown to reduce atmospheric CO₂levels may be included in the Kyoto emission reduction targets. The European Union is committed to a reduction in CO₂ emissions to 92% of baseline (1990) levels during the first commitment period (2008–2012). We have shown recently that there are a number of agricultural land-management changes that show some potential to increase the carbon sink in agricultural soils and others that allow alternative forms of carbon mitigation (i.e. through fossil fuel substitution), but the options differ greatly in their potential for carbon mitigation. The changes examined were, (a) switching all animal manure use to arable land, (b) applying all sewage sludge to arable land, (c) incorporating all surplus cereal straw, (d) conversion to no-till agriculture, (e) use of surplus arable land to de-intensify 1/3 of current intensive crop production (through use of 1/3 grass/arable rotations), (f) use of surplus arable land to allow natural woodland regeneration, and (g) use of surplus arable land for bioenergy crop production. In this paper, we attempt for the first time to assess other (non-CO₂) effects of these land-management changes on (a) the emission of the other important agricultural greenhouse gases, methane and nitrous oxide, and (b) other aspects of the ecology of the agroecosystems. We find that the relative importance of trace gas fluxes varies enormously among the scenarios. In some such as the sewage sludge, woodland regeneration and bioenergy production scenarios, the inclusion of trace gases makes only a small (<10%) difference to the CO₂-C mitigation potential. In other cases, for example the no-till, animal manure and agricultural de-intensification scenarios, trace gases have a large impact, sometimes halving or more than doubling the CO₂-C mitigation potential. The scenarios showing the greatest increase when including trace gases are those in which manure management changes significantly. In the one scenario (no-till) where the carbon mitigation potential was reduced greatly, a small increase in methane oxidation was outweighed by a sharp increase in N₂O emissions. When these land-management options are combined to examine the whole agricultural land area of Europe, most of the changes in mitigation potential are small, but depending upon assumptions for the animal manure scenario, the total mitigation potential either increases by about 20% or decreases by about 10%, shifting the mitigation potential of the scenario from just above the EU's 8% Kyoto emission reduction target (98.9 Tg C y⁻¹) to just below it. Our results suggest that (a) trace gas fluxes may change the mitigation potential of a land management option significantly and should always be considered alongside CO₂-C mitigation potentials and (b) agricultural management options show considerable potential for carbon mitigation even after accounting for trace gas fluxes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Impact of H+ ion irradiation on Matrimid®. I. Evolution in chemical structure

Ion beam irradiation can be used to modify the structure and gas transport properties of glassy polymers. This is the first of two studies that focus on the impact of H⁺ ion irradiation on the structure and permeation properties of the polyimide Matrimid®. Specifically, the evolution in chemical structure after H⁺ irradiation over a range of fluences was analyzed using FTIR spectroscopy and dissolution studies. Although H⁺ ion irradiation at very low ion fluences induced little modification in the chemical structure, irradiation at relatively high ion fluences resulted in crosslinking of the irradiated films. The branched structure of the aliphatic methyl (CH₃) was the most sensitive to the H⁺ ion irradiation. The para‐disubstituted aromatic ring showed the strongest resistance toward ion irradiation and required fairly high doses to induce degradation. Two potential crosslinking mechanisms related to the degradation of the aliphatic methyl and the benzophenone carbonyl were presented. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 2010–2019, 2003