Role of the tubulin-microtubule system in lymphocyte activation

The role of the tubulin-microtubule system was examined in human peripheral blood leukocytes after activation with phytohemagglutinin (PHA). Soluble tubulin and microtubules were measured with a [(3)H]colchicine-binding assay. It was found that the tubulin content of PHA-activated lymphocytes was consistently increased relative to total protein content after 36 h of culture. There was no increase in the proportion of total tubulin synthesis which was present as microtubules at 36 h. Nevertheless, as a result of increased tubulin synthesis, there was a two-to three-fold increase in total microtubular mass. Colchicine, which disrupts microtubles, was used to assess the role of microtubule assembly in the sequence of events which follow lymphocyte activation, namely lymphokine release, protein synthesis, RNA synthesis, and DNA synthesis. Colchicine consistently inhibited DNA synthesis but did not inhibit release of the lymphokine, osteoclast activating factor (OAF). Protein and RNA syntheses were inhibited much less than DNA synthesis. The fact that some effects of PHA on lymphocytes appear to require intact microtubules and at least one does not suggest that the microtubule dependent step in PHA-stimulated lymphocyte activation occurs at a stage after propagation of the signal from the membrane to the cell interior.     

Effects of ischemia on epicardial deformation in the passive rabbit heart

BACKGROUND: Surgically induced ischemia in the arrested heart can result in changes in the mechanical properties of the myocardium. Regions of ischemia may be characterized based on the amount of epicardial deformation for a given load. Computer aided speckle interferometry (CASI), which tracks the movement of clusters of particles, is developed as a technique for measuring epicardial deformation, thereby determining the perfusion status of the passive heart. MATERIALS AND METHODS: Silicone carbide particles and retroreflective beads were dispersed randomly onto the epicardial surface of 11 isolated rabbit hearts to form speckle images. The hearts were arrested with hyperkalemic Krebs-Henseleit buffered solution. Each heart was then exposed to a series of intracavitary pressures, and at each pressure speckle images were acquired with a charge-coupled device (CCD) camera. Nine hearts were exposed to global ischemia, and two hearts were exposed to regional ischemia by occluding the second diagonal branch of the left anterior descending artery (LAD). The hearts were again loaded and the speckle images were acquired. CASI was used to determine the distribution of deformation field. RESULTS: CASI was able to determine displacements with a spatial resolution of about 50 microns. Global ischemia resulted in a significant increase in the maximum principle strain and the first invariant of the 2-D strain tensor. In the regionally ischemic heart, a large difference in deformation between the ischemic and perfused regions was clearly observed. CONCLUSION: Based on epicardial deformation, CASI is able to distinguish between perfused and ischemic myocardium, with a spatial resolution of 50 microns.     

Isoform-specific stimulation of cardiac Na/K pumps by nanomolar concentrations of glycosides

It is well-known that micromolar to millimolar concentrations of cardiac glycosides inhibit Na/K pump activity, however, some early reports suggested nanomolar concentrations of these glycosides stimulate activity. These early reports were based on indirect measurements in multicellular preparations, hence, there was some uncertainty whether ion accumulation/depletion rather than pump stimulation caused the observations. Here, we utilize the whole-cell patch-clamp technique on isolated cardiac myocytes to directly measure Na/K pump current (I(P)) in conditions that minimize the possibility of ion accumulation/depletion causing the observed effects. In guinea pig ventricular myocytes, nanomolar concentrations of dihydro-ouabain (DHO) caused an outward current that appeared to be due to stimulation of I(P) because of the following: (1) it was absent in 0 mM [K(+)](o), as was I(P); (2) it was absent in 0 mM [Na(+)](i), as was I(P); (3) at reduced [Na(+)](i), the outward current was reduced in proportion to the reduction in I(P); (4) it was eliminated by intracellular vanadate, as was I(P). Our previous work suggested guinea pig ventricular myocytes coexpress the alpha(1)- and alpha(2)-isoforms of the Na/K pumps. The stimulation of I(P) appears to be through stimulation of the high glycoside affinity alpha(2)-isoform and not the alpha(1)-isoform because of the following: (1) regulatory signals that specifically increased activity of the alpha(2)-isoform increased the amplitude of the stimulation; (2) regulatory signals that specifically altered the activity of the alpha(1)-isoform did not affect the stimulation; (3) changes in [K(+)](o) that affected activity of the alpha(1)-isoform, but not the alpha(2)-isoform, did not affect the stimulation; (4) myocytes from one group of guinea pigs expressed the alpha(1)-isoform but not the alpha(2)-isoform, and these myocytes did not show the stimulation. At 10 nM DHO, total I(P) increased by 35 +/- 10% (mean +/- SD, n = 18). If one accepts the hypothesis that this increase is due to stimulation of just the alpha(2)-isoform, then activity of the alpha(2)-isoform increased by 107 +/- 30%. In the guinea pig myocytes, nanomolar ouabain as well as DHO stimulated the alpha(2)-isoform, but both the stimulatory and inhibitory concentrations of ouabain were approximately 10-fold lower than those for DHO. Stimulation of I(P) by nanomolar DHO was observed in canine atrial and ventricular myocytes, which express the alpha(1)- and alpha(3)-isoforms of the Na/K pumps, suggesting the other high glycoside affinity isoform (the alpha(3)-isoform) also was stimulated by nanomolar concentrations of DHO. Human atrial and ventricular myocytes express all three isoforms, but isoform affinity for glycosides is too similar to separate their activity. Nevertheless, nanomolar DHO caused a stimulation of I(P) that was very similar to that seen in other species. Thus, in all species studied, nanomolar DHO caused stimulation of I(P), and where the contributions of the high glycoside affinity alpha(2)- and alpha(3)-isoforms could be separated from that of the alpha(1)-isoform, it was only the high glycoside affinity isoform that was stimulated. These observations support early reports that nanomolar concentrations of glycosides stimulate Na/K pump activity, and suggest a novel mechanism of isoform-specific regulation of I(P) in heart by nanomolar concentrations of endogenous ouabain-like molecules.     

Simulated brain tumor growth dynamics using a three-dimensional cellular automaton

We have developed a novel and versatile three-dimensional cellular automaton model of brain tumor growth. We show that macroscopic tumor behavior can be realistically modeled using microscopic parameters. Using only four parameters, this model simulates Gompertzian growth for a tumor growing over nearly three orders of magnitude in radius. It also predicts the composition and dynamics of the tumor at selected time points in agreement with medical literature. We also demonstrate the flexibility of the model by showing the emergence, and eventual dominance, of a second tumor clone with a different genotype. The model incorporates several important and novel features, both in the rules governing the model and in the underlying structure of the model. Among these are a new definition of how to model proliferative and non-proliferative cells, an isotropic lattice, and an adaptive grid lattice.     

A revision of Aceratherium blanfordi Lydekker, 1884 (Mammalia: Rhinocerotidae) from the Early Miocene of Pakistan: postcranials as a key

Rhinocerotids are particularly abundant and diversified in Neogene deposits of the Indian subcontinent, but their systematics is far from being well defined. Based on the revision of old collections and new findings from the Early Miocene of the Bugti Hills and Zinda Pir, Pakistan, ‘Aceratherium blanfordi Lydekker, 1884’ is a chimera, consisting of two dentally convergent but postcranially distinct rhinocerotid taxa: Pleuroceros blanfordi and Mesaceratherium welcommi sp. nov. Postcranial features appear to be much more diagnostic than craniodental morphology in this case. A phylogenetic analysis based on 282 morphological characters scored for 28 taxa (four outgroups and ingroup including both taxa of interest and a ‘branching group’) strengthens this statement and supports Pleuroceros and Mesaceratherium as monophyletic genera within Rhinocerotinae. Both genera are recognized for the first time outside Europe. In the Bugti Hills, P. blanfordi and M. welcommi are part of an exceptionally diversified rhinocerotid fauna, with up to nine species associated in the same locality (Kumbi 4f). This rhinocerotid assemblage confirms the earliest Miocene age (Agenian/Aquitanian) of the upper member of the Chitarwata Formation as a whole. Coeval homotaxic rhinocerotid faunas from Europe (France, Czech Republic) and East Africa (Uganda, Kenya) support broad and sustainable rhinocerotid interchanges amongst South Asia, Europe, and Africa under compatible environmental conditions throughout earliest Miocene times. © 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160 , 139–194.     

Future climate‐driven shifts in distribution of Calanus finmarchicus

Calanus finmarchicus is a key‐structural species of the North Atlantic polar biome. The species plays an important trophic role in subpolar and polar ecosystems as a grazer of phytoplankton and as a prey for higher trophic levels such as the larval stages of many fish species. Here, we used a recently developed ecological niche model to assess the ecological niche (sensu Hutchinson) of C. finmarchicus and characterize its spatial distribution. This model explained about 65% of the total variance of the observed spatial distribution inferred from an independent dataset (data of the continuous plankton recorder survey). Comparisons with other types of models (structured population and ecophysiological models) revealed a clear similarity between modeled spatial distributions at the scale of the North Atlantic. Contemporary models coupled with future projections indicated a progressive reduction of the spatial habitat of the species at the southern edge and a more pronounced one in the Georges Bank, the Scotian Shelf and the North Sea and a potential increase in abundance at the northern edge of its spatial distribution, especially in the Barents Sea. These major changes will probably lead to a major alteration of the trophodynamics of North Atlantic ecosystems affecting the trophodynamics and the biological carbon pump.     

Validation study of a method for assaying DE-310, a macromolecular carrier conjugate containing an anti-tumor camptothecin derivative, and the free drug in human plasma by HPLC and LC/MS/MS

DE-310 is a macromolecular carrier conjugate containing an anti-tumor camptothecin derivative, DX-8951, conjugated to a water-soluble polymer by means of a peptide spacer. New assay methods have been developed to determine the polymer-bonded DX-8951 conjugate, free DX-8951, and Glycyl-DX-8951 in human plasma. Solid-phase extraction was used to extract free DX-8951 and Glycyl-DX-8951 from plasma, and LC/MS/MS (Method I) was used to determine the amount of each analyte. Protein precipitation was used to extract Conjugated DX-8951, which was then digested with thermolysin. HPLC (Method II) was used to determine the productive compound (Phenylalanyl-Glycyl-DX-8951). The lower limit of quantitation of DX-8951 was 50 pg/ml, of Glycyl-DX-8951 was 80 pg/ml, and of Conjugated DX-8951 was 100 ng/ml (as DX-8951 equivalent). Both methods showed satisfactory sensitivity, precision, and accuracy.     

3D Fluid-Structure Interaction Canine Heart Model with Patch to Quantify Mechanical Conditions for Optimal Myocardium Stem Cell Growth and Tissue Regeneration

Right ventricular (RV) dysfunction is a common cause of heart failure in patients with congenital heart defects and often leads to impaired functional capacity and premature death. Myocardial tissue regeneration techniques are being developed for the potential that viable myocardium may be regenerated to replace scar tissues in the heart or used as patch material in heart surgery. 3D computational RV/LV/Patch models with fluid-structure interactions (FSI) were constructed based on data from a healthy dog heart to obtain local fluid dynamics and structural stress/strain information and identify optimal conditions under which tissue regeneration techniques could achieve best outcome. RV/LV/Patch geometry and blood pressure data were obtained from a dog following established procedures. Four FSI models were used to quantify the influence of different patch materials (Dacron scaffold, treated pericardium) on local environment around the patch area, especially focusing on the thickness and stiffness of the patch. Our results indicated that changes in patch stiffness had little impact on the ejection fraction of the right ventricle because the total patch area was small. However, patch stiffness had huge impact on local RV maximum principal stress (Stress-P1) and strain (Strain-P1) around the patch area. Compared to the no-patch model, patch models had increased Stress-P1 and decreased Strain-P1 values in the patch area. Softer patches were associated with greater stress/strain variations. Thinner patch led to complex local flow environment which may have impact on myocytes seeding and RV remodeling. Our multi-physics RV/LV/Patch FSI model can serve as a useful tool to investigate cellular biology and tissue regeneration under localized flow and structural stress environment.