Spontaneous rhythmogenic capabilities of sympathetic neuronal assemblies in the rat spinal cord slice.

Neuronal networks generating rhythmic activity as an emergent property are common throughout the nervous system. Some are responsible for rhythmic behaviours, as is the case for the spinal cord locomotor networks; however, for others the function is more subtle and usually involves information processing and/or transfer. An example of the latter is sympathetic nerve activity, which is synchronized into rhythmic bursts in vivo. This arrangement is postulated to offer improved control of target organ responses compared to tonic nerve activity. Traditionally, oscillogenic circuits in the brainstem are credited with generating these rhythms, despite evidence for the persistence of some frequencies in spinalized preparations. Here, we show that rhythmic population activity can be recorded from the intermediolateral cell column (IML) of thoracic spinal cord slices. Recorded in slices from 10- to 12-day-old rats, this activity was manifest as 8-22 Hz oscillations in the field potential and was spatially restricted to the IML. Oscillations often occurred spontaneously, but could also be induced by application of 5-HT, α-methyl 5-HT or MK212. These agents also significantly increased the strength of spontaneous oscillations. Rhythmic activity was abolished by TTX and attenuated by application of gap junction blockers or by antagonists of GABA(A) receptors. Together these data indicate that this rhythm is an emergent feature of a population of spinal neurons coupled by gap junctions. This work questions the assumption that sympathetic rhythms are dependent on supraspinal pacemaker circuits, by highlighting a surprisingly strong rhythmogenic capability of the reduced sympathetic networks of the spinal cord slice.

Reconstruction of occupational mercury exposures at a chloralkali plant.

OBJECTIVE: To reconstruct historical workplace exposure to mercury (Hg) from 1956 to 1994 at a large chloralkali factory for use in a current epidemiology study of the factory. METHODS: All job activities of the employees were classified into one of 16 exposure categories, and the dates of changes in the processes were identified. Exposures to Hg for each job category, at each period of the plant's operation, were then reconstructed from several data sources. A job-time period-exposure matrix was created, and the individual exposures of former workers were calculated. Data on exposure to Hg in air were compared with modelled concentrations of Hg in air and data on urinary Hg of the employees. RESULTS: Within an exposure category, concentrations of Hg in air were fairly constant for the first 20 years of the factory's operation, but began to increase in the late 1970s. Employees working in the cell room had the greatest exposures to Hg. The exposure estimates had significant correlations (p<0.001) with the urinary data and were well within the modelled range of concentrations of Hg in air. CONCLUSIONS: The highest exposures occurred from 1987 until the plant closed in early 1994 with some exposure categories having time weighted average exposures to Hg greater than 140 microg/m(3).

Outcome analysis of coronary artery bypass grafting: minimally invasive versus standard techniques.

Minimally invasive coronary artery bypass grafting (MIDCAB) procedures are purported to result in improvements in patient management over standard techniques. A comparative study was performed on risk-stratified patients treated with either technique. Following institutional review board approval, a retrospective random chart review was conducted on 27 MIDCAB and 37 standard coronary artery bypass grafting (CABG) patients who were operated on over a 12-month period at the University of Nebraska Medical Center. Risk stratification was accomplished by dividing the two patient populations, MIDCAB and 'standard', into one of four subgroups based on a preoperative risk score. Risk stratification was achieved by dividing the patient populations into one of four subgroups: good, fair, poor and high risk. Both groups received similar operations and surgical interventions, except for the inclusion of cardiopulmonary bypass (CPB). Approximately 200 parameters were collected and analyzed in the following categories: anthropometric, operative and postoperative outcomes. The MIDCAB group had a significantly lower number of vessels bypassed (2.0+/-0.7 vs 3.4+/-0.9, p < 0.0001). Total postoperative blood product transfusions trended higher in the standard group (6.1+/-12.6 U) when compared to the MIDCAB patients (2.3+/-5.5 U, p < 0.15), although not statistically significant. Postoperative inotrope use was significantly less in the MIDCAB group (19% vs 59%, p < 0.002). Ventilator time in the MIDCAB group was 10.5+/-5.4 h vs 15.0+/-12.3 h in the standard group (p < 0.07). The MIDCAB group had an overall greater length of stay, but was only statistically different within the poor-risk subgroup (12.2+/-10.7 vs 7.5+/-3.9, p < 0.04). The results of this study show that when CPB is not utilized in treating patients undergoing CABG procedures, the benefits in regards to patient outcomes are unclear. This necessitates the need for further work when comparing outcomes for risk-stratified patients.

Cardioplegia flow dynamics in an in vitro model.

The flow of fluids in extracorporeal circuits does not conform to conventional Poiseuille mechanics which confounds calculating cardioplegia (CP) flow distribution. The purpose of this study was to quantify CP flow dynamics in a model simulating coronary atherosclerosis across varying sized restrictions. An in vitro preparation was designed to assess hydraulic fluid movement across paired restrictions of 51, 81 and 98% lumen reductions. Volume data were obtained at variable flow, temperature, viscosity and pressure conditions. CP delivered through 14- and 18-gauge (GA) conduits at 8 degrees C and 100 mmHg infusion pressure revealed that both four to one and crystalloid CP solutions had significantly less total percentage flow through the 14-GA conduit, p < 0.0001 and p < 0.001, respectively. Overall, 4:1 CP exhibited the most favorable fluid dynamics at 8 degrees C in that it delivered the highest percentages of total CP flow through the smaller lumen conduit. At both 8 degrees C and 37 degrees C delivery, blood CP resulted in the least homogeneous fluid distribution at all delivery parameters. The results in relation to blood viscosity indicate that, although the 8 degrees C blood CP had a significantly greater viscosity than 37 degrees C blood CP, it did not produce an effect in fluid distribution. These data show that increasing the cardioplegic solution hematocrit causes an inhomogeneous fluid distribution regardless of delivery temperature or infusion pressure.

Removal of hepatocarcinoma cells from blood via cell washing and filtration techniques.

Utilization of autotransfusion during tumor resection remains controversial due to viability of carcinoma cells remaining in collected blood. The purpose of this study was to evaluate autotransfusion techniques combined with leukocyte depleting filters (LDF) for removal of hepatocarcinoma cells from autotransfusate. An in vitro model was created by contaminating expired human erythrocytes with cultured hepatocarcinoma (HEP G2) cells. Autotransfusion devices evaluated were Cobe BRAT2, Sorin STAT-P, and Fresenius CATS. Autotransfusate collected from varying processing conditions were filtered using the Pall Leukoguard RS or Pall Purecell RCQ LDF. Carcinoma concentrations were quantified via Coulter Counter technology. The CATS exhibited higher concentrations of cancer cells in the autotransfusate prior to washing, a 449% increase. This was significantly higher than either the BRAT2 or STAT-P, 350% and 315% respectively. Post washing HEP G2 concentrations in the BRAT2 were significantly higher than the STAT-P and CATS. Doubled wash volumes removed more HEP G2 cells in all trials, reaching statistical significance only in the CATS. LDF resulted in a significant 75% reduction of HEP G2 cells, with no difference between filters. While combination use of autotransfusion devices and leukocyte depleting filters did result in a product with concentrated hematocrit, no technique removed all hepatocarcinoma cells.

Construction of a directed hammerhead ribozyme library: towards the identification of optimal target sites for antisense-mediated gene inhibition.

Antisense-mediated gene inhibition uses short complementary DNA or RNA oligonucleotides to block expression of any mRNA of interest. A key parameter in the success or failure of an antisense therapy is the identification of a suitable target site on the chosen mRNA. Ultimately, the accessibility of the target to the antisense agent determines target suitability. Since accessibility is a function of many complex factors, it is currently beyond our ability to predict. Consequently, identification of the most effective target(s) requires examination of every site. Towards this goal, we describe a method to construct directed ribozyme libraries against any chosen mRNA. The library contains nearly equal amounts of ribozymes targeting every site on the chosen transcript and the library only contains ribozymes capable of binding to that transcript. Expression of the ribozyme library in cultured cells should allow identification of optimal target sites under natural conditions, subject to the complexities of a fully functional cell. Optimal target sites identified in this manner should be the most effective sites for therapeutic intervention.

Purification of (+)-delta-cadinene synthase, a sesquiterpene cyclase from bacteria-inoculated cotton foliar tissue.

A sesquiterpene cyclase whose activity is induced in a glandless, bacterial blight-resistant line of cotton (Gossypium hirsutum L.) catalyses the conversion of (E,E)-farnesyl diphosphate to (+)-delta-cadinene. This enzyme was purified by a combination of salt-induced phase separation, hydroxylapatite fractionation, hydrophobic interaction and strong anion-exchange chromatography, and denaturing polyacrylamide gel electrophoresis, followed by renaturation with Tween 80. The purified enzyme has a molecular weight of 64-65 kDa, and exhibited a single silver-staining band following electrophoresis in analytical denaturing polyacrylamide gels. Amino acid sequences of three tryptic peptides from the enzyme have been determined and are similar to known sequences in other terpene cyclases from plants.

Detection and differentiation of pectic enzyme activity in vitro and in vivo by capillary electrophoresis of products from fluorescent-labeled substrate.

A sensitive assay is described for the detection of pectate-depolymerizing enzymes using capillary electrophoresis of a fluorescent end-labeled pectate oligomer. The labeled oligomer is allowed to react with the enzyme either in vitro or in vivo, such as inside the intercellular spaces of a cotton cotyledon, and after an appropriate incubation time the products are analyzed by capillary electrophoresis. The site and mode of action of the pectate-depolymerizing activity can be inferred from the products. Both endo- and exopolygalacturonase activity, and lyase activity, were distinguished. Since only the fluorescent oligomer and products from its labeled reducing end are detected, there is no interference from other compounds; only pectic enzyme activity is detected. By this type of analysis we can show that there is considerable endo- and exo-polygalacturonase activity in the intercellular spaces of cotton cotyledons.

An Early Indicator of Resistance in Barley to Russian Wheat Aphid.

During early stages of infestation by Russian wheat aphids (Diuraphis noxia [Mordvilko]; RWAs), barley (Hordeum vulgare L.) leaf cells collapsed and showed autofluorescence in the mesophyll and bundle sheath adjacent to the RWA stylet sheath. The response was visually similar to the hypersensitive cell death response, typical of resistance to microbial pathogens. Resistant barley produced significantly more collapsed, autofluorescent cells (CAC) than did susceptible barley. RWA stylet entry sites and sheath paths also fluoresced, making them easy to observe in whole leaf sections. The number of CAC increased with the number of RWAs and with the number of days of feeding in resistant plants. The CAC could be observed 1 d following infestation, making this the most rapid plant response toward the RWAs known to date. The response may be useful in screening for resistant plants and may provide insight into resistance mechanisms in barley.

Importance of amino acid 101 within capsid protein VP1 for modulation of Theiler's virus-induced disease.

We constructed a Theiler's virus mutant designated DA3304, in which the amino acid at position 101 of VP1 was changed from a threonine to an alanine. Because of this single amino acid change, DA3304 could still produce a biphasic central nervous system disease similar to that produced by the wild-type DA virus. However, DA3304 was significantly attenuated in both the acute and the chronic phases and induced smaller demyelinating lesions than the wild-type DA virus. The data are most compatible with the attenuated phenotype in DA3304 being due to the change of binding efficiency between the virus and receptor resulting from the physical alteration at the mutation site.

HLA-DR and -DQ allelic sequences in multiple sclerosis patients are identical to those found in the general population.

The HLA-DR2/Dw2 haplotype is associated with multiple sclerosis (MS) in the North American Caucasian population. HLA-DRB, -DQA, and -DQB N-terminal domain sequences derived from amplified cDNA in a series of North American Caucasian MS patients were examined to determine if unique or rare class II alleles could be found. In addition, class II allelic sequences were analyzed from clinically discordant, HLA-genoidentical siblings from a multiplex MS family. All alleles observed, whether from HLA-DR2/Dw2 positive or negative individuals, were identical to those most commonly expressed in the general population. These data demonstrate that, if HLA class II truly confers susceptibility to MS, commonly expressed alleles are involved.

Role of T cells in resistance to Theiler's virus infection.

Intracerebral infection of C57BL/10SNJ mice with Theiler's virus results in acute encephalitis with subsequent virus clearance and absence of spinal cord demyelination. In contrast, infection of SJL/J mice results in acute encephalitis, virus persistence, and immune-mediated demyelination. These experiments examined the role of T-cell subsets in the in vivo immune response to Theiler's virus in resistant C57BL/10SNJ mice. Depletion of T-cell subsets with monoclonal antibodies (mAbs) directed at CD3 (pan-T-cell marker), CD4+ (class II-restricted) or CD8+ (class I-restricted) T cells resulted in increased frequency of paralysis and death as a result of acute encephalitis. Neuropathologic studies 10 days after infection demonstrated prominent necrosis, primarily in the pyramidal layer of hippocampus and in the thalamus of mice depleted of T-cell subsets. In immunosuppressed and infected C57BL/10SNJ mice, analysis of spinal cord sections 35 days after infection demonstrated small demyelinated lesions relatively devoid of inflammatory cells even though virus antigen could be detected by immunocytochemistry. Both CD4+ and CD8+ T cells are important in the resistance to infection with Theiler's virus in C57BL/10SNJ mice. However, subsequent spinal cord demyelination, to the extent observed in susceptible mice, depends on the presence of virus antigen persistence and a competent cellular immune response.

Interleukin-1 beta decreases HLA class II expression on a glioblastoma multiforme cell line.

Antigens encoded within the major histocompatibility complex (MHC) are not normally expressed in the central nervous system (CNS), but can be induced by treatment with interferon-gamma (IFN-gamma). Other cytokines released during an inflammatory process can potentially influence MHC expression as well. One cytokine of interest is interleukin-1 (IL-1), an immunoregulatory polypeptide that is produced by macrophages and also by cells in the CNS. In this study, the effect of IL-1 beta on MHC expression in a human glioblastoma multiforme cell line, U-105 MG, has been examined. Treatment of U-105 MG with 10 U IL-1 beta/ml for a period of 5 days resulted in a decrease in constitutive cell surface HLA class II expression and limited the induction of class II by IFN-gamma. This effect was also observed on steady-state levels of class II RNA and could be neutralized with antibodies to IL-1 beta. All class II transcripts examined (HLA-DR, -DQ, and -DP alpha and beta) were affected. Class I expression was only marginally changed by IL-1 beta treatment. A minimal concentration of 1 U IL-1 beta/ml was required to reduce class II expression and a kinetics experiment indicated that U-105 MG must be treated for at least 4 days with IL-1 beta for a decrease in class II expression to be observed. This study suggests that IL-1 may play a role in limiting immunoreactivity in the CNS by limiting class II induction.

Immune response gene products (Ia antigens) on glial and endothelial cells in virus-induced demyelination.

Theiler's murine encephalomyelitis virus induced central nervous system demyelination in susceptible strains of mice with s, q, v, p, and f H-2D alleles. We used immunoelectron microscopy to look for differential production of class II immune response gene products (Ia) within astrocytes, oligodendrocytes, microglia, and endothelial cells. Spinal cord sections from susceptible mice (B10.S and B10.ASR2) showed increased content of Ia in glial and endothelial cells. In contrast, resistant mice [B10.S(9R)] showed minimal Ia production within the CNS. The findings indicate an important role of class II immune response products on glial cells during demyelination after virus infection.

Glucoprivic responsivity after insulin in hypodipsic and in water deficit rats.

Long Evans rats were made acutely hypodipsic through quinine adulteration of their water supply, and they ate normally in response to 2, 4, and 6 unit insulin injections. Other subjects were made chronically hypodipsic through adulteration; and while their glucoprivic feeding was lowered absolutely, it remained relatively elevated over their control session intake. These data indicate that subjects with reduced drinking abilities can respond to the insulin-induced glucoprivic challenge and that hydrational factors apparently do not serve as critical confounds in brain lesion investigations into the glucoprivic feeding response.