Analysis of Intestinal Perfusion Data for Highly Permeable Drugs Using a Numerical Aqueous Resistance—Nonlinear Regression Method

Purpose. To develop, validate and apply a method for analyzing the intestinal perfusion data of highly permeable compounds using the Numerical Aqueous Resistance (NAR) theory and nonlinear regression (NAR-NLR) and to compare the results with the well-established Modified Boundary Layer (MBL) Analysis. Methods. The NAR-NLR method was validated and the results were compared to the MBL analysis results using previously reported cephradine jejunal perfusion data. Using the Single Pass Intestinal Perfusion (SPIP) method, the concentration dependence of intestinal permeability was investigated for formycin B, proline, and thymidine, three compounds reported to be absorbed by carrier-mediated transport processes. The MBL and NAR-NLR analyses were then applied to the three sets of SPIP data. Results. The results demonstrate that the intrinsic MBL transport parameters were highly variable and, in one case, the analyses failed to give a statistically significant Michaelis constant. The MBL mean dimensionless wall permeabilities (P*_w) were greater than the NAR-NLR P*_w and were also highly variable. In all cases, the NAR-NLR variability was significantly lower than the MBL variability. The extreme variability in the MBL-calculated P*_w is due to the sensitivity of P*_w when the fraction of unabsorbed drug (C_m/C_o) is low or, alternatively, when P*_w approached the aqueous permeability, P*_aq. Conclusions. The NAR-NLR method facilitates the analysis of intestinal perfusion data for highly permeable compounds such as those absorbed by carrier-mediated processes at concentrations below their K_m. The method also allows for the use of a wider range of flow conditions than the MBL analysis resulting in more reliable and less variable estimates of intestinal transport parameters as well as intestinal wall permeabilities.     

Morphology of retinal ganglion cells in lungless salamanders (fam. Plethodontidae): an HRP and Golgi study

The family Plethodontidae consists of nearly two-thirds of all living urodeles; most of them possess highly developed visual abilities. We investigated the morphology of retinal ganglion cells (RGCs) in four representative species by means of the horseradish peroxidase method in flatmounts and in transverse sections and with the Golgi method in transverse sections. In flatmount preparations, four classes of RGCs were found, differing in dendritic arborization, dendritic field size, and stratification pattern of dendrites in the inner plexiform layer (IPL). Class-1 cells had small dendritic fields (29-44 microns 2) and arborized throughout the entire depth of the IPL. Class-2 cells had medium to large dendritic fields (75-206 microns 2) and mostly arborized in two or three laminae or in a diffuse fashion in the IPL. Class-3 cells had medium to large dendritic fields (72-200 microns 2) but sparse dendritic arborization. They only arborized in the proximal lamina of the IPL. Class-4 cells had large dendritic fields (273-626 microns 2) and branched in the most sclerad stratum of the IPL. No large differences in intraspecific soma size of the different RGC classes were detected (although interspecific soma size varied to a considerable degree) and no "giant" cells typically found in other vertebrate retinas were present. The results suggest that, with respect to the pattern of arborization and stratification of dendrites, lungless salamanders possess morphological classes of RGC similar to those found in frogs, but the morphology of RGCs in lungless salamanders seems to be simplified in comparison to frog RGCs. This simplification might be a consequence of paedomorphosis.     

The role of pioneer neurons in the development of mouse visual cortex and corpus callosum

The primordial plexiform neuropil is very critical to neocortical development. The pioneer neurons, mainly Cajal-Retzius cells in the marginal zone, and subplate neurons in the subplate, differentiate from the primordial plexiform neuropil. In this study, the development of corpus callosum, visual cortex, and subcortical pathways has been observed in C57BL/6 mice with various methods, such as DiI labeling in vitro and in vivo, DiI and DiA in vitro double labeling, immunocytochemistry, and in vivo BrdU and Fast Blue labeling. As early as E14, the primordial plexiform neuropil can be found in the telencephalic wall, and it contains many pioneer neurons. On E15 the primordial plexiform neuropil differentiates into the marginal zone and the subplate. Cajal-Retzius cells exist in the marginal zone, and subplate neurons are in the subplate. Either Cajal-Retzius cells or subplate neurons have long projections toward the ganglionic eminence, suggesting that they migrate tangentially from the ganglionic eminence. Cajal-Retzius cells are involved in radial migration, and subplate neurons participate to guide pathfinding of subcortical pathways. This study reveals how the pioneer neurons, through radial and tangential migration, play an important role in neocortical formation and in the pathfinding of the corpus callosum and subcortical pathways. Furthermore, DiI labeling in vivo has demonstrated the presence of pioneer neurons all along the corpus callosum pathway, especially in the midline. This suggests that pioneer neurons may also play a role in guiding the pathfinding of the corpus callosum. Accepted: 31 July 2001     

Activity in deep intermediate layer collicular neurons during interrupted saccades

The activity of neurons located in the deep intermediate and adjacent deep layers (hereafter called just deep intermediate layer neurons) of the superior colliculus (SC) in monkeys was recorded during saccades interrupted by electrical stimulation of the brainstem omnipause neuron (OPN) region. The goal of the experiment was to determine if these neurons maintained their discharge during the saccadic interruption, and thus, could potentially provide a memory trace for the intended movement which ends accurately on target in spite of the perturbation. The collicular neurons recorded in the present study were located in the rostral three-fifths of the colliculus. Most of these cells tended to show considerable presaccadic activity during a delayed saccade paradigm, and, therefore, probably overlap with the population of SC cells called buildup neurons or prelude bursters in previous studies. The effect of electrical stimulation in the OPN region (which interrupted ongoing saccades) on the discharge of these neurons was measured by computing the percentage reduction in a cell's activity compared to that present during non-interrupted saccades. During saccade interruption about 70% of deep intermediate layer neurons experienced a major reduction (30% or greater) in their activity, but discharge recovered quickly after the termination of the stimulation as the eyes resumed their movement to finish the saccade on the target. Therefore, the pattern of activity recorded in most of the deep intermediate layer neurons during interrupted saccades qualitatively resembled that previously reported for the saccade-related burst neurons which tend to be located more dorsally in the intermediate layer. In contrast, some of our cells (30%) showed little or no perturbation in their activity caused by the saccade interrupting stimulation. Because all the more dorsally located burst neurons and the majority of our deep intermediate layer neurons show a total or major suppression in their discharge during interrupted saccades, it seems unlikely that the colliculus by itself could maintain an accurate memory of the desired saccadic goal or the remaining dynamic motor error required to account for the accuracy of the resumed movement which occurs following the interruption. However, it remains possible that the smaller proportion of our neurons whose activity was not perturbed during interrupted movements could play a role in the mechanisms underlying saccade accuracy in the interrupted saccade paradigm. Interrupted saccades have longer durations than normal saccades to the same target. Therefore, we investigated whether the discharge of our deeper collicular cells was also necessarily prolonged during interrupted saccades, and, if so, how the prolongation compared to the prolongation of the saccade. Sixty percent of our sample neurons showed a prolongation in discharge that was approximately the same as the prolongation in saccade duration (difference < 15 ms in magnitude). The, observation that temporal discharge in our neurons was perturbed to roughly match saccadic temporal perturbation suggests that dynamic feedback about ongoing saccadic motion is provided to the colliculus, but does not necessarily imply that this structure is the site responsible for the computation of dynamic motor error.     

阴部外动脉阴茎支皮瓣尿道成形术的应用解剖

目的：为设计阴部外动脉阴茎皮瓣转位尿道成形术提供依据。方法：30侧经动脉内灌注红色乳胶的成人尸体，解剖观测阴部外动脉起始、行程；着重阴部外动脉阴茎支在阴茎的走行、分支分布。结果：阴部外动脉始于股动脉，外径1．8±0．4mm，伴行静脉1-2支，汇入大隐静脉。阴茎支可视为本干的延续，经耻骨结节两侧靠近阴茎，分别经2(10)点、3(9)点和1(11)点进入阴茎，多数分出背侧支、腹侧支分布阴茎皮肤。外径0．8±0．2mm。结论：阴茎皮肤血管恒定，以阴茎支为蒂，可在阴茎外侧或背外侧、腹外侧设计皮瓣，用于尿道成形术。术式已在临床应用，效果满意。     

Renewal of the membrane complex of Schistosoma mansoni is closely associated with lipid metabolism

Metabolic pathways leading to lipid biosynthesis in four different developmental stages of Schistosoma mansoni were explored and quantified by incubation in the presence of labeled precursors in a chemically defined medium. At the schistosomulum stage and in male, female, or paired worms, glycerol and oleate incorporation into neutral lipids, mainly in the form of triacylglycerols, was greater than into phospholipids, whereas in 11-and 15-day-old worms, synthesis mainly led to phospholipids. Incorporation into phospholipids was recovered largely in phosphatidylcholine, and distribution into other phospholipids depended on the developmental stage. Incorporation of choline and ethanolamine into their respective phospholipids represented up to 15% of the parasitic phospholipid content. The formation of phosphatidylcholine by phosphatidylethanolamine methylation occurred mainly in the immature parasitic stages. Inositol incorporation was also measurable, whereas [14C]serine incorporation was low or undetectable. Addition of 1-palmitoyl-2-[14C]oleyl phosphatidylcholine revealed a very high uptake of this phospholipid by the immature stages but further metabolism was not detectable. In contrast, adult S. mansoni were completely unable to take up or absorb this exogenous phospholipid. The most striking aspect of this study was the relatively high metabolic activity in 11-day-old worms and the lower but sustained activity on day 15 and at the schistosomulum stage. By comparison, biosynthetic activity in adult S. mansoni, on which research studies have been focused until now, was very low. We also discuss the participation of lipid metabolism in the constant renewal of the membrane complex which is essential to parasitism by S. mansoni.     

A statistical analysis and comparison of soma diameter spectra for classical neurones from different regions of the cat retinal ganglion cell layer

Multimodal soma diameter spectra for neurones of the cat retinal ganglion cell layer have been represented by three subpopulations of independent, normal diameter distribution. Recurrent computation according to the technique of Vibert and Caille (1978) has extracted best fit populations for samples from various regions of central and peripheral retina. The model subpopulations from all these regions did not differ significantly in their relative proportions or variance. Significant progressive variation between subpopulations representing different regions of retina were observed only in the mean diameter of the and mode cells. The parameters of the mode population were statistically uniform across the retina. The cat retina thus appears to be more homogeneously organized than has been suggested elsewhere.     

New Insights into Lamellar Twisting in Transcrystalline Polyethylene

Summary: The morphology of the transcrystalline layer grown by nucleating high density polyethylene on fibers of ultra high molecular weight polyethylene was investigated by microbeam synchrotron X‐ray diffraction. Scanning with a 2 micron step size, it was possible to determine that near the fiber surface, the polymer chains of the transcrystalline layer are oriented at an angle of approx. 41° with respect to the fiber axis. This is consistent with the lamellar fold surface (the {201} plane) being close to perpendicular to the fiber axis. The X‐ray data support gradual twisting of the lamellae about the growth direction (the orthorhombic crystallite b‐axis) at a rate of ～0.85° per micron of radial distance from the fiber surface.

Polarized light micrograph of the transcrystalline layer in a PE/PE composite. The width of the fiber is approximately 20 μm.     

Real-time imaging of individual fluorescent-protein color-coded metastatic colonies in vivo

We have established stable, bright green fluorescent protein (GFP)- or red fluorescent protein (RFP)-expressing HT-1080 human fibrosarcoma clones. These cell lines showed similar cell proliferation rates and high-frequency experimental lung metastasis. The HT-1080-GFP and -RFP clones enable simultaneous real-time dual-color imaging in the live animal. HT-1080 cells were transduced with retroviral vectors containing GFP or RFP and the neomycin resistance gene. Stable transformants were selected stepwise with G418 up to 800 μl/ml. Subsequently, high GFP- or RFP-expressing clones, HT-1080-GFP or HT-1080-RFP, respectively, were selected. 3×10⁶ cells from each clone were mixed and injected into the tail vein of SCID mice. The cells seeded the lung at high frequency with subsequent formation of pure green and pure red colonies as well as mixed yellow colonies with different patterns visualized directly on excised lungs. The lung metastases were also visualized by external fluorescence imaging in live animals through skin-flap windows over the chest wall. Lung metastases were observed on the lung surface of all mice. SCID mice well tolerated multiple surgical procedures for direct-view imaging via skin-flap windows. Real-time metastatic growth of the two different colored clones in the same lung was externally imaged with resolution and quantification of green, red, or yellow colonies in live animals. The color coding enabled determination of whether the colonies grew clonally or were seeded as a mixture with one cell type eventually dominating, or whether the colonies grew as a mixture. The simultaneous real-time dual-color imaging of metastatic colonies described in this report gives rise to the possibility of color-coded imaging of clones of cancer cells carrying various forms of gene of interest. This revised version was published online in July 2006 with corrections to the Cover Date.