Odor, drug and toxin analysis with neuronal networks in vitro: extracellular array recording of network responses

Neurons, by virtue of intrinsic electrophysiological mechanisms, represent transducers that report the dynamics of cell death, receptor-ligand interactions, alterations in metabolism, and generic membrane perforation processes. In cell culture, mammalian neurons form fault-tolerant, spontaneously active systems with great sensitivity to their chemical environment and generate response profiles that are often concentration- and substance-specific. Changes in action potential patterns are usually detected before morphological changes and cell damage occur, which provides sensitivity and reversibility. Such biological systems can be used to screen rapidly for novel pharmacological substances, toxic agents, and for the detection of certain odorants. Existing simple culture preparations can already be employed effectively for the detection of chemical compounds. So far, three strategies have been investigated in pilot experiments: (1) Substance-dependent major changes in spontaneous native activity patterns. All synaptically active agents (e.g. glutamate, strychnine, N-methyl D-aspartic acid) as well as metabolic poisons generate such changes. (2) Substance-dependent changes in network oscillations via disinhibition. The regularized, oscillatory activity is altered by synaptically and metabolically active substances, ion channel blockers, and toxins. (3) Detection of paroxysmal responses indicating major, pathological membrane currents in large subpopulation of cells. We have explored these three strategies via 64 channel array recordings using spontaneously active murine spinal cord cultures. The glycine receptor blocker strychnine reliably generated increased multichannel bursting at 5-20 nM and regular, coordinated bursting above 5 microM. During biculline-induced network oscillations many compounds alter oscillation frequencies or terminate activity in a substance-specific manner. Finally, the gp120 protein of the AIDS virus (at 1 microgram/ml) produces massive, unique paroxysmal discharges that may last as long as 2 min. These results indicate that cultured neuronal networks are practical systems that can be used for the detection and identification of a great variety of chemical substances. The concept of dynamic fingerprinting to identify specific compounds is discussed.     

The satellite RNA of barley yellow dwarf virus-RPV is supported by beet western yellows virus in dicotyledonous protoplasts and plants

The subgroup II luteovirus barley yellow dwarf virus-RPV (BYDV-RPV) acts as a helper virus for a satellite RNA (satRPV RNA). The subgroup II luteovirus beet western yellows virus (BWYV) and the ST9-associated RNA (ST9a RNA), a BWYV-associated RNA that encodes a polymerase similar to those of subgroup I luteoviruses, were assayed for their ability to support replication of satRPV RNA. SatRPV RNA was replicated in tobacco protoplasts in the presence of BWYV RNA or a mixture of BWYV plus the ST9a RNA, but not in the presence of ST9a RNA alone. ST9a RNA stimulated BWYV RNA accumulation which, in turn, increased the accumulation of satRPV RNA. SatRPV RNA was encapsidated in BWYV capsids primarily as circular monomers, which differs from the linear monomers found in BYDV (RPV + PAV) particles. SatRPV RNA was transmitted to Capsella bursa-pastoris plants by aphids only in the presence of BWYV and ST9a RNA. SatRPV RNA reduced accumulation of both BWYV helper and ST9a nonhelper RNAs in plants but did not affect symptoms. The replication of satRPV RNA only in the presence of subgroup II luteoviral RNAs but not in the presence of RNAs with subgroup I-like polymerase genes, in both monocotyledonous and dicotyledonous hosts, suggests that the specificity determinants of satRPV RNA replication are contained within the polymerase genes of supporting viruses rather than in structural genes or host plants.     

A temporal study of TPN-induced changes in gut-associated lymphoid tissue and mucosal immunity

BACKGROUND: Total parenteral nutrition (TPN) is associated with decreases in small-intestinal gut-associated lymphoid tissue (GALT) T cells, B cells, and IgA levels and impairs IgA-mediated defenses in the respiratory tract. The impaired respiratory tract defenses are speculated to be due to reduced respiratory tract IgA levels. OBJECTIVES: To determine the time course of GALT cell reductions and document any changes in respiratory tract IgA levels in mice receiving TPN. DESIGN: Prospective randomized trial. SETTING: Animal research laboratory. MATERIALS: Thirty-five male ICR mice weighing 25 to 35 g. INTERVENTIONS: Mice underwent cannulation with intravenous catheters and received chow for 2 days followed by TPN for 0 (n=6), 1 (n=6), 2 (n=6), 3 (n=6), 4 (n=6), or 5 (n=5) days. Mice were killed after receiving TPN their respective number of days. The small intestine was harvested, and washings were obtained from the small intestine and the respiratory tract. Lymphocytes and IgA levels were analyzed by flow cytometry and enzyme-linked immunosorbent assay, respectively. MAIN OUTCOME MEASURES: Lymphocyte yields from Peyer patches, intraepithelial spaces, and the lamina propria; IgA levels from the small intestine and the respiratory tract. RESULTS: T- and B-cell yields in the Peyer patches and lamina propria were significantly reduced by day 2 (P<.05) and thereafter compared with day 0. The lamina propria CD4+/CD8+ ratio declined significantly by day 4 (P<.05) compared with day 0. Small-intestinal and respiratory tract IgA levels were significantly diminished by day 3 (P<.05) and thereafter compared with day 0. CONCLUSION: Total parenteral nutrition produces rapid changes in GALT cell profiles and reduces respiratory tract IgA levels consistent with the impairment of respiratory IgA-mediated defenses.     

Efficacy of measles vaccine during the 1993 measles epidemic in Korea

Immunization to eliminate measles is recommended at 15 months of age with the option of giving vaccine at 6 to 9 months of age during measles outbreaks in Korea. Because of the recent resurgence of measles and concern about the possibility of reduced vaccine efficacy caused by genomic differences between vaccine virus and contemporary wild measles viruses, we conducted a measles vaccine efficacy study involving children with household exposure ages 1 to 5 years during measles outbreak that had occurred 1993 in Seoul and Seong-nam city, with the demographic analysis of patients brought to the hospitals. A total of 380 patients (M:F = 216:164) were included in this study. Two hundred nine cases (55.0%) occurred in children less than 5 years of age, and 167 (43.9%) were younger than 16 months of age. The recorded age-specific incidence rates showed bimodal patterns, i.e. highest peak in those below 16 months of age and second peak in those ages 6 to 9 years of age. Only 9.6% (16 of 167) of the measles cases less than 16 months, 59.5% (25 of 42) of those 16 months to 4 years and 91.8% (157 of 171) of the cases in school age children have been vaccinated. Attack rates among the 122 vaccinated siblings and 12 unvaccinated siblings ages 1 to 5 years who contacted measles were 5.7 and 75%, respectively, and the clinical vaccine efficacy was 92.4% (95% confidence interval, 83.6, 96.4). The high vaccine efficacy in household exposures suggests that measles outbreaks in Korea are not caused by reduced vaccine efficacy.     

Prognostic values of echocardiography compared with clinical variables in suspected heart disease. Multivariate analysis of long-term prognosprognosis in 456 patients

The aim of the study was to evaluate the prognostic significance of clinical and echocardiographic data in patients referred for echocardiography in a retrospective analysis. Four hundred and fifty-six patients from a district hospital were studied. Survival after three years was 64%. Multivariate analysis identified five factors with independent prognostic information (relative risks of death are shown in brackets): left ventricular wall motion index (WMI) < or = 1.2 by echocardiography (2.5), status as in-patient (2.1), age > 65 years (1.7), clinical heart failure (1.9) and atrial fibrillation (1.5). When information on age, hospitalisation status, heart failure and heart rhythm had already been entered in the Cox model, echocardiographic results such as decreased WMI and dilated right ventricle still gave further prognostic information. We conclude that among conventional clinical and echocardiographic data WMI was the strongest predictor of long-term survival, and, despite prior knowledge of major clinical features, echocardiography provided further prognostic information.     

The infrared dichroism of transmembrane helical polypeptides

Polarized attenuated total internal reflectance techniques were applied to study the infrared dichroism of the amide I transition moment in two membrane-bound peptides that are known to form oriented transmembrane helices: gramicidin A in a supported phospholipid monolayer and Ac-Lys2-Leu24-Lys2-amide (L24) in oriented multibilayers. These studies were performed to test the ability of these techniques to determine the orientation of these peptides, to verify the value of optical parameters used to calculate electric field strengths, to examine the common assumptions regarding the amide I transition moment orientation, and to ascertain the effect of surface imperfections on molecular disorder. The two peptides exhibit marked differences in the shape and frequency of their amide I absorption bands. Yet both peptides are highly ordered and oriented with their helical axes perpendicular to the membrane surface. In the alpha-helix formed by L24, there is evidence for a mode with type E1 symmetry contributing to amide I, and the amide I transition moment must be more closely aligned with the peptide C=O (< 34 degrees) than earlier studies have suggested. These results indicate that long-standing assumptions about the orientation of amide I in a peptide require some revision, but that in general, infrared spectroscopy yields reliable information about the orientation of membrane-bound helical peptides.