Influence of Ca2+ depletion on cytoskeleton and nucleolus morphology in Trypanosoma brucei.

Trypanosoma brucei bloodstream forms were incubated in a calcium-free medium containing 10 microM ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). Under these conditions, addition of 5 microM calcium ionophore A23187 led to striking morphological alterations, as judged by light and electron microscopy. The cytoskeleton of trypanosomes consists of a subpellicular corset of microtubules. Characteristically four of these microtubules are attached invariantly to an extension of the endoplasmic reticulum at the flagellar attachment site. Specifically in this area calcium depletion led to the polymerization of additional microtubules and to a retraction of the endoplasmic reticulum extension from its usual position. Additionally, A23187 led to nucleolus segregation, as revealed by immunocytochemistry using antibodies against DNA and fibrillarin, respectively. Nucleolus segregation, but not microtubule accumulation, was also obtained by using 20 microM camptothecin, a specific inhibitor of topoisomerase I. Our data suggest that intracellular calcium regulation might be important for specific depolymerization/polymerization reactions during the course of cell division and the formation of functional ribosomes.     

A Retraining Program for Inactive Physicians

During the past two years a pilot project was conducted in which 19 inactive physicians were retrained in preparation for resumption of active practice. The initial program consisted of a flexible training program of six months to one year patterned after conventional internship-residency concepts. During the second year the program was modified by providing an initial condensed indoctrination period of two months'' duration especially designed for this purpose, followed by a preceptorship type of training.The project was considered successful in permitting trainees to enter some form of active medical work, or to enroll in formal specialty training. The observations made by the faculty of the program and its accomplishments are discussed in the light of the effort expended and the cost of the project.     

Differences in hyaluronic acid-mediated functions and signaling in arterial, microvessel, and vein-derived human endothelial cells

Hyaluronic acid (HA), a nonsulfated glycosaminoglycan, regulates cell adhesion and migration. Small HA fragments (3-25 disaccharide units) induce neovascularization. We investigated the effect of HA and a HA fragment (10-15 disaccharide units, F1) on primary human endothelial cells (ECs). Human pulmonary ECs (HPAEC) and lung microvessel ECs (HMVEC-L) bound HA (K(d) approximately 1 and 2.3 nm, respectively) and expressed 17,780 and 16,690 HA binding sites, respectively. Both ECs showed HA-mediated cell adhesion; however, HMVEC-L was 1.5-fold better. Human umbilical vein ECs neither bound HA nor showed HA-mediated adhesion. All three ECs expressed CD44 ( approximately 110 kDa). The expression of receptor for HA-mediated motility (RHAMM) (approximately 80 kDa) was the highest in HMVEC-L, followed by HPAEC and human umbilical vein ECs. RHAMM, not CD44, bound HA in all three ECs. F1 was better than HA and stimulated a 2. 5- and 1.8-fold mitogenic response in HMVEC-L and HPAEC, respectively. Both HA and F1 induced tyrosine phosphorylation of p125(FAK), paxillin, and p42/44 ERK in HMVEC-L and HPAEC, which was blocked by an anti-RHAMM antibody. These results demonstrate that RHAMM is the functional HA receptor in primary human ECs. Heterogeneity exists among primary human ECs of different vascular origins, with respect to functional HA receptor expression and function.     

Trypanothione Reductase: A Target Protein for a Combined In Vitro and In Silico Screening Approach

With the goal to identify novel trypanothione reductase (TR) inhibitors, we performed a combination of in vitro and in silico screening approaches. Starting from a highly diverse compound set of 2,816 compounds, 21 novel TR inhibiting compounds could be identified in the initial in vitro screening campaign against T. cruzi TR. All 21 in vitro hits were used in a subsequent similarity search-based in silico screening on a database containing 200,000 physically available compounds. The similarity search resulted in a data set containing 1,204 potential TR inhibitors, which was subjected to a second in vitro screening campaign leading to 61 additional active compounds. This corresponds to an approximately 10-fold enrichment compared to the initial pure in vitro screening. In total, 82 novel TR inhibitors with activities down to the nM range could be identified proving the validity of our combined in vitro/in silico approach. Moreover, the four most active compounds, showing IC₅₀ values of <1 μM, were selected for determining the inhibitor constant. In first on parasites assays, three compounds inhibited the proliferation of bloodstream T. brucei cell line 449 with EC₅₀ values down to 2 μM.     

Cytoskeletal changes correlated with the loss of neuronal polarity in axotomized lamprey central neurons

Axotomy within 500 μm of the soma (close axotomy) causes identified neurons (anterior bulbar cells or ABCs) in the lamprey hindbrain to lose their normal polarity and regenerate axons ectopically from dendritic tips, while axotomy at more distal sites (distant axotomy) results in orthotopic axonal regeneration from the axon stump. We performed immunocytochemical, electron microscopic and in situ hybridization analyses comparing ABCs subjected to close and distant axotomy to elucidate the mechanism by which neuronal polarity is lost. We show that polarity loss in ABCs is selectively and invariably preceded and accompained by the following cellular changes: (1) a loss of many dendritic microtubules and their replacement with neurofilaments, (2) a loss of immunostaining for acetylated tubulin in the soma and proximal dendrites, and (3) an increase of immunostaining for phosphorylated neurofilaments in the distal dendrites. We also show that these changes do not depend on either the upregulation or spatial redistribution of neurofilament message, and thus must involve changes in the routing of neurofilament protein within axotomized ABCs. We conclude that close axotomy causes dendrites to undergo axonlike changes in the mechanisms that govern the somatofugal transport of neurofilament protein, and suggest that these changes require the reorganization of dendritic microtubules. We also suggest that the bulbous morphology and lack of f-actin in the tips of all regenerating sprouts supports the possibility that axonal regeneration in the lamprey CNS does not involve actin-mediated "pulling" of growth cones, but depends instead on the generation of internal extrusive forces.     

Differentiation and visualization of diverse cellular phenotypic responses in primary high-content screening

High-throughput screening, based on subcellular imaging, has become a powerful tool in lead discovery. Through the generation of high-quality images, not only the specific target signal can be analyzed but also phenotypic changes of the whole cell are recorded. Yet analysis strategies for the exploration of high-content screening results, in a manner that is independent from predefined control phenotypes, are largely missing. The approach presented here is based on a well-established modeling technique, self-organizing maps (SOMs), which uses multiparametric results to group treatments that create similar morphological effects. This report describes a novel visualization of the SOM clustering by using an image of the cells from each node, with the most representative cell highlighted to deploy the phenotype described by each node. The approach has the potential to identify both expected hits and novel cellular phenotypes. Moreover, different chemotypes, which cause the same phenotypic effects, are identified, thus facilitating "scaffold hopping."     

NVX-412, a New Oncology Drug Candidate,Induces S-Phase Arrest and DNA Damage in Cancer Cells in a p53-Independent Manner

The new molecular entity quinoxalinhydrazide derivative NVX-412 was identified as a promising drug candidate for the treatment of various cancer types due to its strong cytotoxic activity and relative specificity. Here, we provide first data about the mechanisms of action of NVX-412. We show that NVX-412 exerts its anti-neoplastic activity in a p53-independent manner and induces S-phase arrest and DNA damage as assessed by γH2AX staining. We suggest a bi-modal (dose-dependent) mode of action of NVX-412, being primarily cytostatic at lower and predominantly cytotoxic at higher concentrations. Based on the broad and consistent anti-neoplastic activity observed, NVX-412 holds promise as an effective drug candidate for the treatment of various cancer types, especially for hematological malignancies with highly unmet medical need.