Spatial Analysis of Slowly Oscillating Electric Activity in the Gut of Mice Using Low Impedance Arrayed Microelectrodes

Smooth and elaborate gut motility is based on cellular cooperation, including smooth muscle, enteric neurons and special interstitial cells acting as pacemaker cells. Therefore, spatial characterization of electric activity in tissues containing these electric excitable cells is required for a precise understanding of gut motility. Furthermore, tools to evaluate spatial electric activity in a small area would be useful for the investigation of model animals. We thus employed a microelectrode array (MEA) system to simultaneously measure a set of 8×8 field potentials in a square area of ∼1 mm². The size of each recording electrode was 50×50 µm², however the surface area was increased by fixing platinum black particles. The impedance of microelectrode was sufficiently low to apply a high-pass filter of 0.1 Hz. Mapping of spectral power, and auto-correlation and cross-correlation parameters characterized the spatial properties of spontaneous electric activity in the ileum of wild-type (WT) and W/W^v mice, the latter serving as a model of impaired network of pacemaking interstitial cells. Namely, electric activities measured varied in both size and cooperativity in W/W^v mice, despite the small area. In the ileum of WT mice, procedures suppressing the excitability of smooth muscle and neurons altered the propagation of spontaneous electric activity, but had little change in the period of oscillations. In conclusion, MEA with low impedance electrodes enables to measure slowly oscillating electric activity, and is useful to evaluate both histological and functional changes in the spatio-temporal property of gut electric activity.     

The influence of motor unit composition and stature on fractionated patellar reflex times in untrained men

The purpose of the present study was to investigate the influence of muscle fibre composition and stature on fractionated patellar reflex times in ten healthy untrained men (mean age: 23.3 years, SD 3.1; mass: 65.9 kg, SD 8.5; height: 172.3 cm, SD 5.3). Biopsies were taken from the right vastus lateralis muscle. Using staining for myofibrillar adenosine triphosphatase after pre-incubation at pH 4.3 and 4.6, muscle fibres were classified into slow twitch (ST), fast twitch, oxidative-glycolytic (FTa) and fast twitch, glycolytic (FTb) fibres. Total patellar reflex time (TRT) and its fractionated components--reflex latency (LAT) and reflex motor time (MT)--were obtained from the mean of ten trials in each subject whilst performing Jendrassik's maneuvre. The TRT, LAT and MT were 77.7 ms, SD 16.5, 23.4 ms, SD 1.3 and 54.2 ms, SD 16.3, respectively. The LAT was significantly correlated to the percentage number of ST (r = 0.758, P less than 0.05) and FTa fibres (r = -0.657, P less than 0.05), fast twitch:slow twitch ratio (r = -0.799, P less than 0.01) and to the height of the subjects (r = 0.901, P less than 0.001), whereas TRT and MT were not significantly correlated with either fibre types or the height of the subjects. From these results it can be concluded that the LAT during the patellar reflex is influenced by muscle fibre composition and the length of the sensory and/or motor nerve.     

Stimulation of T lymphocyte proliferation by monoclonal antibodies against GD3 ganglioside

Cell-surface gangliosides are presumed to play a role in cell growth and differentiation. With the use of monoclonal antibodies directed against GD3, a disialoganglioside expressed predominantly by cells of neuroectodermal origin, we have found that GD3 is expressed by a subpopulation of cells of the immune system including: 1) fetal thymocytes in subcortical regions and near vessels, 2) lymph node lymphocytes in interfollicular areas and near vessels, and 3) a small subset of T cells in the peripheral blood. Mouse monoclonal antibodies (two IgGs, one IgM, and F(ab')2 fragments) reacting with GD3 were found to stimulate proliferation of T cells derived from peripheral blood. Proliferation of T cells was observed even in cultures depleted of macrophages, suggesting that activation by anti-GD3 was not dependent on the presence of accessory cells. T cell proliferation was maximum between days 5 and 7 of stimulation and was preceded by expression of interleukin 2 receptors. No stimulation was observed with control antibodies of the identical isotype or with monoclonal antibodies recognizing the gangliosides GD2 or GM2. During stimulation by anti-GD3 monoclonal antibodies, there was an expansion of the GD3+ pool of T cells, but depletion of GD3+ T cells prior to stimulation abrogated the response. Proliferation induced by binding to GD3 could be augmented by exogenous interleukin 2 and phytohemagglutinin. Anti-CD3 (T3) monoclonal antibodies had little or no effect. These results demonstrate that binding to GD3 on the surface of T cells can elicit signals for T cell proliferation.     

Orosomucoid (ORM) typing by isoelectric focusing: evidence for gene duplication of ORM1 and genetic polymorphism of ORM2

Summary It has been demonstrated that the genetic polymorphism of human serum orosomucoid (ORM) is controlled by polymorphic ORM1 and monomorphic ORM2 loci. In this study a Japanese family was encountered in which several members had puzzling electrophoretic patterns consisting of four bands. The ORM patterns were due to the products of a duplicated ORM1 locus haplotype (ORM1 ^* 2·1) or the products of new variant alleles at the ORM2 locus. The ORM1 ^* 2·1 haplotype is very common in the Japanese population, occurring at an allele frequency of 0.16. The increased occurrence of ORM1 2-1 and the heterogeneity in band intensity among ORM1 2-1 phenotypes could be explained in terms of a duplicated gene ORM1 ^* 2·1. The ORM2 locus proved to be polymorphic, with six alleles in the Japanese population. Dedicated to Professor Dr. K. Nishigami on the occasion of his 60th birthday     

Comparative assessment of the resistance of the unstirred water layer to solute transport between two different intestinal perfusion systems

The resistance of the unstirred water layer to solute transport was estimated in two different intestinal single-pass perfusion systems for a comparative study, using D-glucose as a model compound. One is a well established perfusion system in anesthetized rats as a standard (system A). The other is the one in unanesthetized rats for comparison (system B). It was demonstrated that in system B as well as in system A the resistance of the unstirred water layer to D-glucose transport should be taken into account and this resistance, accordingly, the effective thickness of the unstirred water layer (delta) which is assumed to be in proportion to its resistance, could be described as a function of the perfusion rate by using a film model. The delta decreased with increasing perfusion rate and was larger in system A than in system B at each perfusion rate; 785 microns in system A versus 319 microns in system B at the perfusion rate of 0.16 ml/min and 337 microns versus 184 micron at that of 2.95 ml/min. Thus in system B the effective thickness, accordingly, the resistance, of the unstirred water layer was reduced to about 50% of that in system A, but the resistance of the unstirred water layer could still account for 85% of the total resistance at the maximum as far as D-glucose absorption was concerned, while 93% in system A. These results suggest that, compared with perfusion experiments in anesthetized rats (system A), the resistance of the unstirred water layer is reduced but cannot be left out of consideration even if perfusion experiments are performed in unanesthetized rats (system B). And the lower resistance of the unstirred water layer in system B was attributed to a turbulent flow in contrary to a laminar flow in system A.     

Determination of kinetic parameters of a carrier-mediated transport in the perfused intestine by two-dimensional laminar flow model: effects of the unstirred water layer

The kinetic parameters of a carrier-mediated transport for D-glucose and for taurocholate were determined from rat in situ intestinal single perfusion experiments. The true parameters were obtained by the two-dimensional laminar flow model, in which the solute concentration at the aqueous-intestinal membrane interface can be calculated numerically without assuming the aqueous diffusion layer, discriminating the effects of the unstirred water layer. The true Michaelis constant was 4.5 mM for D-glucose and 1.5 mM for taurocholate. The true maximal transport velocity was 3.4 nmol/s per cm2 for D-glucose and 0.29 nmol/s per cm2 for taurocholate. The apparent Michaelis constant was raised by the factor of 6.6 for D-glucose and 3.6 for taurocholate due to the effects of the unstirred water layer. The maximal transport velocity was relatively unaffected by the unstirred water layer in both compounds. The values of the effective (operational) thickness of the unstirred water layer were compatible with those reported previously by employing various experimental methods. The kinetic parameters obtained in vitro everted sacs, for comparison, almost coincided with the true ones in situ. Therefore, the two-dimensional laminar flow model is shown to be valid not only for determining the kinetic parameters of a carrier-mediated transport in situ but also for predicting the absorption rate in situ from the uptake rate in vitro.     

Determination of the membrane permeability coefficient and the reflection coefficient by the two-dimensional laminar flow model for intestinal perfusion experiments

We performed single perfusion experiments in the small intestine of rats in order to prove that the two-dimensional laminar flow model is suitable to determine the membrane permeability coefficient and the reflection coefficient. We used progesterone as an aqueous-diffusion-limited drug, urea as a membrane transport-limited drug and the tritiated water as an intermediate substance. The membrane permeability coefficient for progesterone was calculated to be 3.6 X 10(-4) cm/s. This value did not change when the thickness of the aqueous diffusion layer was altered by increasing the perfusion rate 10-fold. It was directly demonstrated that the two-dimensional laminar flow model was suitable to analyze the data of intestinal perfusion experiments. Membrane permeability coefficients for urea and tritiated water were determined to be 3.4 X 10(-5) cm/s and 8.9 X 10(-5) cm/s, respectively. In the presence of water absorption with the hypotonic perfusion solution, the reflection coefficient for urea was 0.84. This value is thought to be theoretically reasonable, suggesting the usefullness of the two-dimensional laminar flow model to obtain the reflection coefficient in the intestinal membrane.     

Intracellular localization of alkaline phosphatase in freshly isolated foetal rat hepatocytes

Summary The cytochemical localization of alkaline phosphatase activity in foetal rat hepatocytes was examined in relation to the pattern of cell to cell attachment during cell isolation and culture. In foetal hepatocytesin vivo, alkaline phosphatase was exclusively localized on the bile canalicular membrane. In freshly isolated foetal hepatocytes, however, the activity was present in the endoplasmic reticulum, nuclear envelope, Golgi apparatus, tubulo-vesicular organelles, and over the entire plasma membrane. In monolayer cells cultured for one or two days, the activity was localized on the reconstituted bile canalicular membrane, plasma membrane sites adjacent to neighbouring cells and on the bottom surface of the monolayer, but was detected in none of the intracellular organelles. Biochemical alkaline phosphatase activity did not change during isolation of the cells. These results suggest that, in foetal hepatocytes, loss of cell—cell contact may induce a temporal disturbance, or dedifferentiation, in their membrane system.     

5C6-F4, a novel 100,000-dalton rat lymphocyte activation antigen defined by monoclonal antibody

Con A-activated rat thymocytes were used to immunize mice, and immune spleen cells were fused with NS/1 myeloma cells. One clone, designated 5C6-F4, reacted strongly with Con A-activated rat thymocytes and some LPS-activated rat spleen cells but not with normal thymocytes, spleen cells, or bone marrow cells of rat origin. The 5C6-F4 did not react with Con A-activated thymocytes of mouse origin. Immunoprecipitation of 5C6-F4 antigen from surface-iodinated Con A-activated rat thymocytes or LPS-activated rat spleen cells revealed its m.w. to be approximately 100,000. The kinetic studies of the expression of 5C6-F4 antigen revealed that 5C6-F4 antigen was detectable at 6 hr after Con A stimulation of rat spleen cells, whereas IL 2 receptor (IL 2R) was detectable at 12 hr. The appearance of 5C6-F4 antigen and IL 2R precede the onset of DNA synthesis of Con A-activated spleen cells. Thus, 5C6-F4 antigen is classified as early activation antigen. The 5C6-F4 inhibits the lymphocyte proliferation induced by mitogen and the IL 2-driven rat T cell proliferation. Sequential immunoprecipitation study as well as binding inhibition study indicated that the 5C6-F4 antigen is distinct from IL 2R molecule. The 5C6-F4 antigen appears to be a novel rat lymphocyte activation antigen that exhibits immunoregulatory function and also may serve as a useful marker of T cell activation.