Whole-cell and single-channel alpha1 beta1 gamma2S GABAA receptor currents elicited by a "multipuffer" drug application device

Pharmacological characterization of ion channels and receptors in cultured neurons or transfected cell lines requires microapplication of multiple drug solutions during electrophysiological recording. An ideal device could apply a large number of solutions to a limited area with rapid arrival and removal of drug solutions. We describe a novel "multipuffer" rapid application device, based on a modified T-tube with a nozzle made from a glass micropipette tip. Drug solutions are drawn via suction from open reservoirs mounted above the recording chamber through the device into a waste trap. Closure of a solenoid valve between the device and the waste trap causes flow of drug solution though the T-tube nozzle. Any number of drug solutions can be applied with rapid onset (50-100 ms) after a brief fixed delay (100-200 ms). Recombinant alpha1beta1gamma2S GABAA receptors (GABARs) transfected into L929 fibroblasts were recorded using whole-cell and single-channel configurations. Application of GABA resulted in chloride currents with an EC50 of 12.2 microM and a Hill slope of 1.27, suggesting more than one binding site for GABA. GABAR currents were enhanced by diazepam and pentobarbital and inhibited by bicuculline and picrotoxin. Single-channel recordings revealed a main conductance state of 26-28 pS. This device is particularly suitable for rapid, spatially controlled drug applications onto neurons or other cells recorded in the whole-cell configuration, but is also appropriate for isolated single-channel or multichannel membrane patch recordings.     

Discrete-time tracking systems incorporating Lur'e plants with multiple non-linearities

The concept of absolute tracking (Gruji? and Porter 1980) is extended so as to embrace digital Lur'e control systems subjected to multiple unmeasurable disturbances and multiple unmeasurable command inputs. It is shown that the discrete-time tracking characteristics of closed-loop digital control systems incorporating Lur'e plants with multiple non-linearities can be established on the basis of the discrete-time absolute stability characteristics of associated free systems. These general results are illustrated by synthesizing a discrete-time tracking system incorporating a Lur'e plant with two non-linearities.     

Phase-structure analysis of brass by anodic linear-sweep voltammetry

Anodic linear-sweep voltammetry (ALSV) was applied to electrodeposited alloy layers and to metallurgically obtained Cu–Zn alloys of different composition and structure. Metallurgically obtained samples covered the range of composition in which , (+), and (+) intermediate phases were detected by X-ray. The ALSV of samples containing less than 30 wt % Zn exhibited a single peak at –0.08 to –0.1 V/SCE before massive dissolution, starting at about –0.05 V/SCE. The presence of the -phase gave another peak at a significantly more negative potential (–0.25 to –0.35 V/SCE). The ALSV of electrodeposited alloys were significantly more complex than the former, depending on the deposition potentials, with peaks attributable to pure Zn, to the -phase, to the -phase as well as to the -phase, present in most cases and dissolving at potentials similar to that of pure Cu. ALSV was shown to be a reliable and practical method for a fast determination of both the composition and the phase structure of electrodeposited brass.     

In situ ESEM study of the thermal decomposition of chrysotile asbestos in view of safe recycling of the transformation product

The thermal transformation of asbestos into non-hazardous crystalline phases and their recycling is a promising solution for the "asbestos problem". The most common asbestos-containing industrial material produced worldwide is cement-asbestos. Knowledge of the kinetics of thermal transformation of asbestos fibers in cement-asbestos is of paramount importance for the optimization of the firing process at industrial scale. Here, environmental scanning electron microscopy (ESEM) was used for the first time to follow in situ the thermal transformation of chrysotile fibers present in cement-asbestos. It was found that the reaction kinetics of thermal transformation of chrysotile was highly slowed down in the presence of water vapor in the experimental chamber with respect to He. This was explained by chemisorbed water on the surface of the fibers which affected the dehydroxylation reaction and consequently the recrystallization into Mg-silicates. In the attempt to investigate alternative and faster firing routes for the decomposition of asbestos, a low melting glass was mixed with cement-asbestos and studied in situ to assess to which extent the decomposition of asbestos is favored. It was found that the addition of a low melting glass to cement-asbestos greatly improved the decomposition reaction and decreased the transformation temperatures.     

Ultrastructure and biochemistry of thyroid carcinoma

Thirty-two thyroid tumors, 9 benign, 23 malignant, and 12 samples of normal thyroid tissue were examined by light and electron microscopy. Thyroglobulin content was also measured in the tissues and, in a limited number of cases, enzymatic activities were determined, such as thyroid peroxidase-iodinase, acid protease, and deiodinase. The presence of significant amounts of 19S, 27S and 12S thyroglobulin was well correlated with the ability of the tumors to accumulate radioiodine. It is suggested that the presence of thyroglobulin be used as a marker of potential function of thyroid carcinoma. Two types of ultrastructural changes were observed in thyroid carcinoma. The first one was interpreted as accompanying the progressive loss of function of thyroid tumors, and was represented by the modifications of highly specialized structures such as RER, lysosomal dense bodies, colloid, etc. The second one is suspected to reflect the malignant transformation of the follicular cell. This concerned namely the nuclei, mitochondria, and intracytoplasmic inclusions. These changes may have a diagnostic value since they were not observed in benign conditions.     

A comparative study of treadmill tests and heel raising exercise for peripheral arterial disease

OBJECTIVES: This two part study validated a 1 min treadmill exercise test and compared this with simple heel raising exercise. METHODS: In an initial study of 24 claudicants (aged 43-79, median 63 years), ankle pressures were measured immediately after repeated treadmill exercises: for 1 min, until onset of claudication, and until maximum tolerated walking distance. Absolute value, fall and percent change in pressures were calculated. The results of this part of the study were then used as a "gold standard" for comparison with 30 s of heel raising and treadmill exercise. This second stage was performed on 21 symptomatic limbs (14 claudicants aged 42-73, median 69 years). RESULTS: Variability was least for pressures expressed as percent change after 1 min of exercise. The paired t-test revealed a significant correlation between the two methods of exercise (p < 0.05). CONCLUSION: Heel raising produced changes in ankle pressure which correlated well with those induced by treadmill exercise. We recommend the use of simple heel raising when a stress test is required to diagnose lower limb arterial insufficiency in the outpatient clinic.     

Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA

A quantitative molecular technique was developed for rapid analysis of microbial community diversity in various environments. The technique employed PCR in which one of the two primers used was fluorescently labeled at the 5' end and was used to amplify a selected region of bacterial genes encoding 16S rRNA from total community DNA. The PCR product was digested with restriction enzymes, and the fluorescently labeled terminal restriction fragment was precisely measured by using an automated DNA sequencer. Computer-simulated analysis of terminal restriction fragment length polymorphisms (T-RFLP) for 1,002 eubacterial sequences showed that with proper selection of PCR primers and restriction enzymes, 686 sequences could be PCR amplified and classified into 233 unique terminal restriction fragment lengths or "ribotypes." Using T-RFLP, we were able to distinguish all bacterial strains in a model bacterial community, and the pattern was consistent with the predicted outcome. Analysis of complex bacterial communities with T-RFLP revealed high species diversity in activated sludge, bioreactor sludge, aquifer sand, and termite guts; as many as 72 unique ribotypes were found in these communities, with 36 ribotypes observed in the termite guts. The community T-RFLP patterns were numerically analyzed and hierarchically clustered. The pattern derived from termite guts was found to be distinctly different from the patterns derived from the other three communities. Overall, our results demonstrated that T-RFLP is a powerful tool for assessing the diversity of complex bacterial communities and for rapidly comparing the community structure and diversity of different ecosystems.     

Quantitative analysis of acute axonal pathology in experimental spinal cord contusion

The major sensorimotor deficits that result from traumatic spinal cord injury (SCI) are due to loss of axons in ascending and descending pathways of the white matter (WM). Experimental treatments administered after a standardized SCI can reduce WM loss and long-term functional deficits. Thus, a significant proportion of WM loss occurs secondary to the mechanical injury and may be a target for therapeutic intervention. Presently, we know little of how and when secondary injury mechanisms operate in the WM after SCI. We therefore used a standardized rat model of clinically relevant contusion injury to examine axonal pathology over the first 24 h by light and electron microscopy. Based on qualitative evaluation of tissue at 15 min, 4 h, and 24 h after a "mild" SCI produced with a weight-drop device (10 g x 2.5 cm), we selected areas from the ventromedial WM at the lesion epicenter for quantitative analyses. We compared axon number and the proportion of axons with various axoplasmic and myelin abnormalities over time after SCI, as well as the effect of axon size on degree of pathology and loss. We found by 4 h postinjury (pi) axonal pathology was more severe than at 15 min and that a significant loss of large diameter axons had occurred; no significant additional loss of axons was seen by 24 h pi. When we compared axonal pathology after a more severe contusion (10 g x 17.5 cm), we found a greater loss of axons at 4 h. In addition, a higher proportion of the remaining axons demonstrated pathological alterations. We developed a semi-quantitative Axonal Injury Index (AII) as an overall measure of axonal pathology that was sensitive to the effects of injury severity at 4 h pi. The AII has greater statistical power than our individual measures of axonal pathology. Our results suggest that it may be possible to use the AII at 4 h pi to assess effects of potential therapeutic agents on acute axonal pathology after SCI.