Ethanol effects on nitric oxide production in cerebral pial cultures.

BACKGROUND: Although alcohol abusers are known to have higher incidences of hemorrhagic cerebrovascular diseases, it is not known whether these changes are associated with ethanol (EtOH) action on nitric oxide (NO) production in the cerebrovascular cells. The purpose of this study was to examine the effects of EtOH treatment on basal and cytokine-induced NO production in cortical pial cultures. METHODS: Cell cultures for this study included murine primary pial vascular cells, primary glial cells and cortical neurons. These cells were exposed to cytokines or EtOH for 24 to 48 hr. The culture media were used for measurement of nitrite, as an indication for NO release, and lactate dehydrogenase (LDH), as an index of cell membrane integrity. In addition, immunocytochemical determinations were carried out to identify cell types and to assess inducible nitric oxide synthase (iNOS). RESULTS: Exposure of primary pial vascular cultures to cytokines that consisted of interleukin-1 beta (IL-1 beta; 250 pg/mL) and interferon-gamma (IFNgamma; 2 ng/mL) or to EtOH (50 to 100 mM) for 24 to 48 hr significantly elevated NO production. NO production could be attenuated by N-nitro-L-arginine (N-arg), a nonspecific NOS inhibitor, or aminoguanidine (AG), an iNOS inhibitor. Increased iNOS immunoreactivity was observed in cytokines- or EtOH-treated pial cells. When pial cells were cocultured with cortical neurons, prolonged EtOH exposure led to a large increase in NO production as well as LDH release. However, this increase was not observed in pial culture alone or in mixed cortical culture. Nevertheless, inhibition of NO production with N-arg or AG did not alter the EtOH-induced LDH release in the pial cells cocultured with cortical neurons. CONCLUSION: These results show that EtOH exposure led to increased production of NO in primary pial cell culture. In mixed culture that contained cortical neurons and pial cells, EtOH induced increase in NO as well as LDH release, which is an indication of loss of cell membrane integrity. However, EtOH-mediated LDH release in mixed cortical pial cultures was not a consequence of the increase in NO production by these cells. Studies that use mixed cortical-pial cultures may provide a unique in vitro system for examining the interactions among glial cells, neurons, and cerebrovascular cells.

Disaster coordination and management: summary and action plans.

INTRODUCTION: Disaster is a collective responsibility requiring coordinated response from all parts of society. This theme focused on coordination and management issues in a diverse range of scenarios. METHODS: Details of the methods used are provided in the preceding paper. The chairs moderated all presentations and produced a summary that was presented to an assembly of all of the delegates. Although the main points developed in Themes 1 and 4 were different from each other (as reported in the Results section), their implementation was similar. Therefore, the chairs of both groups presided over one workshop that resulted in the generation of a set of Action Plans that then were reported to the collective group of all delegates. RESULTS: The main points developed during the presentations and discussions included: (1) the need for evidence-based assessments and planning, (2) the need for a shift in focus to health-sector readiness, (3) empowerment of survivors, (4) provision of relief for the caregivers, (5) address the incentives and disincentives to attain readiness, (6) engage in joint preparation, response, and training, (7) focus on prevention and mitigation of the damage from events, and (8) improve media relations. There exists a need for institutionalization of processes for learning from experiences obtained from disasters. DISCUSSION: Action plans presented include: (1) creation of an Information and Data Clearinghouse on Disaster Management, (2) identification of incentives and disincentives for readiness and develop strategies and interventions, and (3) act on lessons learned from evidence-based research and practical experience. CONCLUSIONS: There is an urgent need to proactively establish coordination and management procedures in advance of any crisis. A number of important insights for improvement in coordination and management during disasters emerged.

Education issues in disaster medicine: summary and action plan.

INTRODUCTION: Change must begin with education. Theme 8 explored issues that need attention in Disaster Medicine education. METHODS: Details of the methods used are provided in the introductory paper. The chairs moderated all presentations and produced a summary that was presented to an assembly of all of the delegates. The chairs then presided over a workshop that resulted in the generation of a set of action plans that then were reported to the collective group of all delegates. RESULTS: Main points developed during the presentations and discussion included: (1) formal education, (2) standardized definitions, (3) integration, (4) evaluation of programs and interventions, (5) international cooperation, (6) identifying the psychosocial consequences of disaster, (7) meaningful research, and (8) hazard, impact, risk and vulnerability analysis. DISCUSSION: Three main components of the action plans were identified as evaluation, research, and education. The action plans recommended that: (1) education on disasters should be formalized, (2) evaluation of education and interventions must be improved, and (3) meaningful research should be promulgated and published for use at multiple levels and that applied research techniques be the subject of future conferences. CONCLUSIONS: The one unanimous conclusion was that we need more and better education on the disaster phenomenon, both in its impacts and in our response to them. Such education must be increasingly evidence-based.

Acute exposure to trichloroethylene differentially alters the susceptibility to chemoconvulsants in mice.

The effects of a common industrial solvent, trichloroethylene (TCE), which was once used as an anesthetic agent but its in vivo mechanism is still unknown, on convulsant-induced seizures in mice were examined. Pretreatment with TCE (250-2000 mg/kg, i.p.) significantly increased pentylenetetrazol (PTZ)-, picrotoxin (PIC)-, bicuculline (BIC)-, strychnine (STY)-, 4-aminopyridine (4-AP)- and N-methyl-D-aspartate (NMDA)-induced convulsion thresholds and lethal doses. However, the increase in convulsion thresholds and lethal doses was much greater for GABAergic antagonists (PIC, BIC, and PTZ) than non-GABAergic convulsants (STY, 4AP, and NMDA) following 2000 mg/kg TCE administration. Pre-treatment of mice with disulfiram (an inhibitor of CYP 4502E1) but not 4-methyl pyrazole (an inhibitor of alcohol dehydrogenase) significantly prolonged the time required for TCE (5000 mg/kg, i.p.) to induce the loss of righting reflex. These results suggest that acute exposure to TCE differentially alters the susceptibility to chemically induced convulsions in mice. The anticonvulsive effect of TCE may be predominantly mediated by GABA(A) receptors. In addition, TCE appears to exert a direct anesthetic effect.

Characterization of calcitonin-containing liposome formulations for intranasal delivery.

Calcitonin-containing liposome formulations were characterized to obtain information for evaluation of their feasibility in intranasal delivery. The parameters of liposomal charge characteristics, charge inducing agent concentration, calcitonin concentration and pH of the medium on the loading efficiency and leakage behaviour, and the chemical stability of calcitonin in liposomes were investigated. Results showed that the loading efficiency of calcitonin increased with increasing the added concentration of calcitonin. The magnitude of the loading efficiency due to the liposomal charge of negative, positive and neutral characteristics was in the order of negatively charged liposome > neutral liposome > positively charge liposome. The increase of molar ratio of phosphatidylserine in liposomes showed an increase of loading efficiency; while, the increase of molar ratios of stearylamine showed a decrease of loading efficiency. The loading efficiency at pH 7.4 was greater than that at pH 4.3. The leakage of positively charged liposomes was greater than that of neutral and negatively charged liposomes. The leakage at pH 4.3 was faster than that at pH 7.4. The leakage of positively charged liposomes increased as temperature increased. The chemical stability of calcitonin in both solution and liposomes demonstrated a pseudo-first-order kinetic degradation. Less degradation was observed at pH 3.4 and 4 degrees C. The degradation rate of calcitonin in solution, or in positively charged, negatively charged, and neutral liposomes, exhibited no significant difference. The particle size of the calcitonin-containing liposomes after storage for 1 month at pH 4.3 and 4 degrees C showed little change.

Adsorption of calcitonin to glass.

Surface adsorption of calcitonin on soda lime silica glass was investigated. An attempt was also made to examine the effect of additives on the inhibition of calcitonin adsorption. Results showed that the adsorption isotherms were of the Langmuir and Freundlich type, depending on pH. Less adsorption was found for calcitonin at pH 4.3. The addition of nonionic surfactants such as Pluronic F68 and Tween 80 to the calcitonin solutions demonstrated inhibition of absorption and reduction of adsorption rate. The addition of chlorobutanol also showed the effect of minimizing adsorption.

The motion control of a statically stable biped robot on an uneven floor.

This work studies the motion control of a statically stable biped robot having seven degrees of freedom. Statically stable walking of the biped robot is realized by maintaining the center-of-gravity inside the convex region of the supporting foot and/or feet during both single-support and double-support phases. The main points of this work are framing the stability in an easy and correct way, the design of a bipedal statically stable walker, and walking on sloping surfaces and stairs.

Molecular contacts between nebulin and actin: cross-linking of nebulin modules to the N-terminus of actin.

Nebulin, a giant actin binding protein, coextends with actin and is thought to form a composite thin filament in the skeletal muscle sarcomere. To understand the molecular interactions between nebulin and actin, we have applied chemical cross-linking techniques to define molecular contacts between actin and ND8, a two-module nebulin fragment that promotes actin polymerization and inhibits depolymerization by binding to both G- and F-actin. The formation of a 1:1 complex with a dissociation constant of 4.9 microM between ND8 and G-actin was demonstrated by fluorescence titration of dansyl-ND8 with G-actin. Treatment with a zero-length cross-linker, l-ethyl-3-[3-(dimethylamino) propyl]carbodiimide (EDC), cross-linked the ND8-G-actin complex covalently without impairing actin's ability to polymerize. End-labeling Western blot and sequence and mass analyses of purified conjugated peptides revealed the cross-linking between lysine 5 of ND8 and the two N-terminal acidic residues of G-actin. Similarly, we have shown by end-labeling that cross-linking of ND8 to F-actin occurred at the N-terminus of actin protomer. The binding of nebulin to the N-terminus of actin is likely to be significant in its ability to affect actin polymerization. Furthermore, the association of nebulin modules with the actin N-terminus in subdomain 1 supports the hypothesis that nebulin wraps around the outer edges of actin filaments where Sl, tropomyosin, and several actin binding proteins are known to interact.

Nebulin as an actin zipper. A two-module nebulin fragment promotes actin nucleation and stabilizes actin filaments.

Nebulin is a family of giant muscle proteins (700-900 kDa) that interact with actin to form composite thin filaments in the skeletal muscle sarcomere. This modular protein is composed predominantly of repeating sequence modules of 31-38 residues. To understand the minimum size and number of sequence modules that are required for actin interaction, we studied the behavior of a highly soluble two-module nebulin fragment ND8 that was expressed in Escherichia coli. By fluorescence spectroscopy with pyrenyl-actin and co-sedimentation assays, we observed the following. 1) ND8 greatly accelerated actin nucleation, especially in a buffer that is suboptimal for actin nucleation. The presence of ND8 abolished the lag phase of actin polymerization and increased the net extent of steady state polymerization, thereby reducing the critical concentration of actin polymerization. 2) ND8 reduced the rate of actin depolymerization and might increase the rate of elongation. 3) Cytochalasin E, which caps both ends of actin filaments, inhibited the effect of ND8 on actin polymerization and caused the depolymerization of actin-ND8 complexes. These data suggest that ND8 interacts with actin in such a fashion that it stabilizes the actin nuclei and slows the depolymerization from the ends of actin filaments. 4) The binding stoichiometry of ND8 to F-actin, as estimated by co-sedimentation assays, is 1 to 2 mol of ND8 to 1 mol of actin with an apparent dissociation constant of 20 to 40 microM. Our data suggest that nebulin-actin interaction promotes actin nucleation and stabilizes preformed actin filaments, both of which are desirable attributes of a length-regulating template for actin filaments of the skeletal muscle. Each nebulin molecule may contain as many as 100-200 actin binding domains to form a zipper-like nebulin/actin composite filament.

Quantitative determination of high molecular weight alkaline phosphatase in patients with colorectal cancer by polyacrylamide gel electrophoresis.

We present a new method for the quantitative determination of high molecular weight alkaline phosphatase (ALP). This method consists of electrophoretic separation on polyacrylamide, incubation of the ALP isoenzymes with substrate, gel drying and quantitative evaluation by densitometric measurement. This method separates high molecular weight ALP from the other isoenzymes. Precision of the method is calculated by the coefficient of variation ranging from 1.2 to 9.5% for samples with different high molecular weight ALP values. The diagnostic sensitivity for detecting colorectal cancer is 60%.

Prosodic structure in language understanding: evidence from tone sandhi in Mandarin.

Two experiments show that prosodic information plays a crucial role in the processing of sentences of Standard Mandarin Chinese, where local lexical ambiguities may occur due to the operation of a tone sandhi rule. In Chinese, each word is associated with a tone; in this paper, the term "Mandarin tone sandhi" refers to a phonological rule that changes the first of two consecutive low tones (Tone 3) to a rising tone (Tone 2). As a result, a two-syllable sequence with a rising tone followed by a low tone is ambiguous. In Experiment 1, listeners identified lexical tones for ambiguous, unambiguous, and nonsense words in phrasal contexts where the tone sandhi rule might have applied. Comparable results in the lexical versus nonsense conditions indicate that judgments did not rely simply on lexically stored tonal information, but also made reference to the tonal context of the phrase. In Experiment 2, subjects chose the most likely written English translation for auditory sentences of Mandarin. Global prosodic information was manipulated to create different levels of "prosodic closeness" between two critical items in a tone sandhi environment, while the syntactic relation between these items was held constant. Results show that listeners relied on the prosodic structure of the phrases to determine whether or not the tone sandhi rule had applied, and consequently to identify individual lexical items. The evidence is taken to support the notion that prosodic structure influences auditory language comprehension processes.