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PubMed1998

The C-terminus of c-ABL is required for proliferation and viability signaling in a c-ABL/erythropoietin receptor fusion protein.

Activated ABL oncogenes cause B-cell leukemias in mice and chronic myelogenous leukemia in humans. However, the mechanism of transformation is complex and not well understood. A method to rapidly and reversibly activate c-ABL was created by fusing the extra-cytoplasmic and transmembrane domain of the erythropoietin (EPO) receptor with c-ABL (EPO R/ABL). When this chimeric receptor was expressed in Ba/F3 cells, the addition of EPO resulted in a dose-dependent activation of c-ABL tyrosine kinase and was strongly antiapoptotic and weakly mitogenic. To evaluate the contributions of various ABL domains to biochemical signaling and biological effects, chimeric receptors were constructed in which the ABL SH3 domain was deleted (triangle upSH3), the SH2 domain was deleted (triangle upSH2), the C-terminal actin-binding domain was deleted (triangle upABD), or kinase activity was eliminated by a point mutation, K290M (KD). The mutant receptors were stably expressed in Ba/F3 cells and analyzed for signaling defects, proliferation, viability, and EPO-induced leukemia in nude mice. When compared with the ability of the full-length EPO R/ABL receptor to induce proliferation and support viability in vitro, the triangle upSH3 mutant was equivalent, the triangle upSH2 mutant was moderately impaired, and the triangle upABD and KD mutants were profoundly impaired. None of these cell lines caused leukemia in mice in the absence of pharmacological doses of EPO. However, in mice treated with EPO (10 U/d), death from leukemia occurred rapidly with wild-type and triangle upSH3. However, time to death was prolonged by at least twofold for triangle upSH2 and greater than threefold for triangle upABD. This inducible model of ABL transformation provides a method to link specific signaling defects with specific biological defects and has shown an important role for the C-terminal actin-binding domain in proliferation and transformation in the context of this receptor/oncogene.

Amino Acid Substitution↗

PubMed1999

Synaptic control of spiking in cerebellar Purkinje cells: dynamic current clamp based on model conductances.

Previous simulations using a realistic model of a cerebellar Purkinje cell suggested that synaptic control of somatic spiking in this cell type is mediated by voltage-gated intrinsic conductances and that inhibitory rather than excitatory synaptic inputs are more influential in controlling spike timing. In this paper, we have tested these predictions physiologically using dynamic current clamping to apply model-derived synaptic conductances to Purkinje cells in vitro. As predicted by the model, this input transformed the in vitro pattern of spiking into a different spike pattern typically observed in vivo. A net inhibitory synaptic current was required to achieve such spiking, indicating the presence of strong intrinsic depolarizing currents. Spike-triggered averaging confirmed that the length of individual intervals between spikes was correlated to the amplitude of the inhibitory conductance but was not influenced by excitatory inputs. Through repeated presentation of identical stimuli, we determined that the output spike rate was very sensitive to the relative balance of excitation and inhibition in the input conductances. In contrast, the accuracy of spike timing was dependent on input amplitude and was independent of spike rate. Thus, information could be encoded in Purkinje cell spiking in a precise spike time code and a rate code at the same time. We conclude that Purkinje cell responses to synaptic input are strongly dependent on active somatic and dendritic properties and that theories of cerebellar function likely need to incorporate single-cell dynamics to a greater degree than is customary.

Animals↗

PubMed2005

A theory of marks and mind: the effect of notational systems on hominid brain evolution and child development with an emphasis on exchanges between mothers and children.

A model of human language requires a theory of meaningful marks. Humans are the only species who use marks to think. A theory of marks identifies children's scribbles as significant behavior, while hypothesizing the importance of rotational systems to hominid brain evolution. By recognizing the importance of children's scribbles and drawings in developmental terms as well as in evolutionary terms, a marks-based rather than a predominantly speech-based theory of the human brain, language, and consciousness emerges. Combined research in anthropology, primatology, art history, neurology, child development (including research with deaf and blind children), gender studies and literacy suggests the importance of notational systems to human language, revealing the importance of mother/child interactions around marks and sounds to the development of an expressive, communicative, symbolic human brain. An understanding of human language is enriched by identifying marks carved on bone 1.9 million years ago as observational lunar calendar-keeping, pushing proto-literacy back dramatically. Neurologically, children recapitulate the meaningful marks of early hominins when they scribble and draw, reminding us that literacy belongs to humankind's earliest history. Even more than speech, such meaningful marks played - and continue to play - decisive roles in human brain evolution. The hominid brain required a model for integrative, transformative neural transfer. The research strongly suggests that humankind's multiple literacies (art, literature, scientific writing, mathematics and music) depended upon dyadic exchanges between hominid mothers and children, and that this exchange and sharing of visuo-spatial information drove the elaboration of human speech in terms of syntax, grammar and vocabulary. The human brain was spatial before it was linguistic. The child scribbles and draws before it speaks or writes. Children babble and scribble within the first two years of life. Hands and mouths are proximal on the sensory-motor cortex. Gestures accompany speech. Illiterate brains mis-pronounce nonsense sounds. Literate brains do not. Written language (work of the hands) enhances spoken language (work of the mouth). Until brain scans map the neurological links between human gesture, speech and marks in the context of mother/caregiver/child interactions, and research with literate and illiterate brains document even more precisely the long-term differences between these brains, the evolutionary pressure of marks on especially flexible maternal and infant brain tissue that occurred 1.9 million years, radically changing primate brain capabilities, requires an integrated theory of marks and mind.

Animals↗

PubMed2001

Nonlinear parameters estimation from sequential short time data series.

Procedures of nonlinear parameter estimation require large samples of data. In stationary physiological situations, usually short time series are available. The method of dynamics-dependent windowing and data aggregation procedure are proposed. This technique was tested on chaotic signal generated by Lorenz model and applied to investigate beat-to-beat control of the cardiovascular system in 10 healthy volunteers. Nonivasively recorded blood pressure, respiratory activity and blood oxygen saturation were digitized and saved for further off-line analysis. The experimental procedure consisted of 10 min control--C, 20 voluntary apneas 1 min each-A, interapnea 20 periods of 1 min spontaneous breathing--B, and 10 min free-breathing recovery--R. Respiration signal served as a reference for apnea and interapnea free-breathing identification period. Correlation dimension-CD, according to Grassberger and Procaccia, and recurrence plot strategy, according to Webber and Zbilut, were applied to check dynamical properties of the signals. Results of numerical experiment on Lorenz model, original and transformed by segmentation and aggregation, support our assumption of similarity of their dynamics. Error in CD and recurrence parameters estimation strongly depended on segment length and was about 5% for 600 to 1,200 data points. However, even for segments of 75 to 100 samples, it did not exceed 10% for all, but one, periodic testing signal. Segmentation and aggregation applied to interbeat interval (IBI) and total peripheral resistance (TPR) data showed that CD and recurrence variables estimated separately for apneic and interapneic period and those calculated for mixed (apneic and interapneic) intervals were different. Average CD and recurrence parameters of IBI and TPR for 10 subjects during apnea and interapnea intervals were significantly different than during control and recovery. The lowest CD (mean +/- S.D.) of 6.38 +/- 0.4, 5.62 +/- 0.2 and %recurrence 10.35 +/- 0.8, 6.62 +/- 0.6 (highest ratio 4.95 +/- 0.2, 5.13 +/- 0.3) were observed in apnea for IBI and TPR, respectively. Low values of the estimates computed for mixed periods may suggest the influence of slowly varying, quasiperiodic driving force due to experimental procedure regime. Signal dynamics-dependent windowing and data aggregation regardless of the sequence of data could be a practical solution for nonlinear analysis of very short repeatable time series.

Adult↗

PubMed2001

Mammary glands reconstituted with Neu/ErbB2 transformed HC11 cells provide a novel orthotopic tumor model for testing anti-cancer agents.

The ErbB2 receptor tyrosine kinase (RTK) has been intensely pursued as a cancer therapy target due to its association with breast cancer. In this study we used the HC11 mammary epithelial cell line to develop an orthotopic, ErbB2-driven tumor model for testing efficacy of anti-cancer compounds. HC11 cells were infected with a retrovirus encoding oncogenic NeuT, the rat homolog of ErbB2. Drug-selected populations were introduced into mammary fat pads of Balb/c syngeneic mice cleared of host tissue. The majority of glands injected with HC11-NeuT cells developed mammary tumors which appeared after a 3-4 week latency period and grew rapidly. HC11 cells infected with the control retrovirus showed no tumor growth after injection. Tumor-bearing mice were used to compare the in vivo efficacy of two anti-cancer agents: PKI166, a kinase inhibitor selective for EGF receptor and ErbB2, and Taxol, a microtubule assembly blocker. PKI166 inhibited NeuT-induced mammary tumor growth in a dose-dependent manner and at a dose below the maximum tolerated dose (MTD) was significantly more inhibitory than Taxol at its MTD (57% vs. 25% tumor regression). Importantly, there was a dose-dependent decrease in the phosphotyrosine content of NeuT isolated from PKI166-treated, tumor-bearing mice, providing a mechanistic link between kinase inhibition and its anti-tumor activity. Thus, implantation of genetically manipulated HC11 cells into mammary glands appears to be an excellent model for studying effects of anti-cancer agents in an orthotopic site.

Animals↗

PubMed1998

A dose-dependent transformation of embryotoxicity manifestations in the population of chick embryos.

The world-wide and long-lasting stagnation in general incidence of inborn defects does not agree with the increasing impact of the environment. However, inborn defects alone are definitely a poor indicator of the reproductive risk being accompanied by much more frequent embryotoxicity manifestations -- death of the conceptus and growth retardation. Dose-dependent transformations of the embryotoxicity manifestations may explain both the stagnation in the incidence of inborn defects and the observed shifts in malformation spectra. An attempt was made to design a reliable experimental model to produce individual transformations resulting from the specific dose-response relationships inherited in teratogenesis. A population of chick embryos in different stages of development was exposed to increasing doses of cyclophosphamide, a model teratogen. All types of the transformations were recorded. Prenatal extinction of defective embryos, transition of single moderate defects to more severe and multiple malformations, as well as the changes in the incidence of certain types of defects exhibit a clear-cut positive dose dependence. Introducing this experimental system we will be able to study mechanisms underlying the particular transformations.

Abnormalities, Drug-Induced↗

PubMed2005

Comparison of human and automatic segmentations of kidneys from CT images.

PURPOSE: A controlled observer study was conducted to compare a method for automatic image segmentation with conventional user-guided segmentation of right and left kidneys from planning computerized tomographic (CT) images. METHODS AND MATERIALS: Deformable shape models called m-reps were used to automatically segment right and left kidneys from 12 target CT images, and the results were compared with careful manual segmentations performed by two human experts. M-rep models were trained based on manual segmentations from a collection of images that did not include the targets. Segmentation using m-reps began with interactive initialization to position the kidney model over the target kidney in the image data. Fully automatic segmentation proceeded through two stages at successively smaller spatial scales. At the first stage, a global similarity transformation of the kidney model was computed to position the model closer to the target kidney. The similarity transformation was followed by large-scale deformations based on principal geodesic analysis (PGA). During the second stage, the medial atoms comprising the m-rep model were deformed one by one. This procedure was iterated until no changes were observed. The transformations and deformations at both stages were driven by optimizing an objective function with two terms. One term penalized the currently deformed m-rep by an amount proportional to its deviation from the mean m-rep derived from PGA of the training segmentations. The second term computed a model-to-image match term based on the goodness of match of the trained intensity template for the currently deformed m-rep with the corresponding intensity data in the target image. Human and m-rep segmentations were compared using quantitative metrics provided in a toolset called Valmet. Metrics reported in this article include (1) percent volume overlap; (2) mean surface distance between two segmentations; and (3) maximum surface separation (Hausdorff distance). RESULTS: Averaged over all kidneys the mean surface separation was 0.12 cm, the mean Hausdorff distance was 0.99 cm, and the mean volume overlap for human segmentations was 88.8%. Between human and m-rep segmentations the mean surface separation was 0.18-0.19 cm, the mean Hausdorff distance was 1.14-1.25 cm, and the mean volume overlap was 82-83%. CONCLUSIONS: Overall in this study, the best m-rep kidney segmentations were at least as good as careful manual slice-by-slice segmentations performed by two experienced humans, and the worst performance was no worse than typical segmentations from our clinical setting. The mean surface separations for human-m-rep segmentations were slightly larger than for human-human segmentations but still in the subvoxel range, and volume overlap and maximum surface separation were slightly better for human-human comparisons. These results were expected because of experimental factors that favored comparison of the human-human segmentations. In particular, m-rep agreement with humans appears to have been limited largely by fundamental differences between manual slice-by-slice and true three-dimensional segmentation, imaging artifacts, image voxel dimensions, and the use of an m-rep model that produced a smooth surface across the renal pelvis.

Humans↗

PubMedDate not supplied

TNT phytotransformation pathway characteristics in Arabidopsis: role of aromatic hydroxylamines.

Basic knowledge of the plant transformation pathways of TNT will aid phytoremediation design and assessment. While TNT transformation by plant metabolism has been demonstrated in previous studies, the presence and role of hydroxylamines in the transformation pathway has not been sufficiently understood. Hydroxylamines are unequivocally shown to be formed by plant transformation of TNT by two axenic plant systems (Arabidopsis thaliana and Catharanthusroseus). In addition, confirmation was obtained for conversion of these hydroxylamines to previously identified conjugates. Further characteristics of TNT transformation in Arabidopsis, an increasingly popular model system for genetic and biochemical studies of TNT transformation, were elucidated by [U-14C]TNT mass balance studies and metabolite feeding studies. These studies showed the rapid conversion of TNT to unextractable bound compounds by Arabidopsis seedlings in agreement with the green-liver model. Arabidopsis seedlings formed and transformed 4-substituted metabolites more efficiently than the 2-substituted metabolites. A qualitative kinetic rate analysis of the pathway was performed to propose rate-limiting steps in the pathway and theoretical schemes for improved rates are suggested.

Arabidopsis↗

PubMed1999

Microsomal formation of nitric oxide and cyanamides from non-physiological N-hydroxyguanidines: N-hydroxydebrisoquine as a model substrate.

The microsomal oxidative transformation of a non-physiological N-hydroxyguanidine was demonstrated for the first time for N-hydroxydebrisoquine as a model substrate (Clement et al., Biochem Pharmacol 46: 2249-2267, 1993). The objective of the present work was to further compare this reaction with the analogous oxidation of arginine via N-hydroxyarginine to citrulline and nitric oxide. The oxidation of N-hydroxydebrisoquine by liver microsomes from rats pretreated with dexamethasone not only produced nitric oxide and the urea, but also the cyanamide derivative as the main metabolite. The low stability of the cyanamide derivative, which easily hydrolyzed to the urea derivative, was noted. The formation of all compounds required cosubstrate and the enzyme source. Experiments with catalase, superoxide dismutase, and H2O2 showed that the O2- formed from the enzyme and the substrate apparently participated in the reaction. While the N-hydroxylation of the guanidine involves the usual monooxygenase activity of cytochrome P-450 (Clement et al., Biochem Pharmacol 46: 2249-2267, 1993), the resultant N-hydroxyguanidine decoupled the monooxygenase. Nitric oxide was detected by the oxyhemoglobin assay. To examine the influence of enzymatically formed nitric oxide on the formation of the metabolites, the N-hydroxydebrisoquine was incubated with SIN-1 as nitric oxide donor under aerobic conditions. It was again possible to detect the cyanamide and urea derivatives, with the latter as main metabolite. It was concluded that the microsomal transformation of N-hydroxydebrisoquine produces a cyanamide and nitric oxide which reacts with N-hydroxydebrisoquine to form the urea derivative. The purely chemical reaction of the unsubstituted N-hydroxyguanidine with nitric oxide gave similar results (Fukuto et al., Biochem Pharmacol 43: 607-613, 1992). In conclusion, similarities (formation of a urea derivative) and differences (formation of a cyanamide derivative) between the physiological oxidation of N-hydroxy-L-arginine by nitric oxide synthases and non-physiological N-hydroxyguanidines by cytochrome P-450 were observed. Furthermore, non-physiological N-hydroxyguanidines can be regarded as nitric oxide donors.

Animals↗

PubMed2004

Learning multiple visuomotor transformations: adaptation and context-dependent recall.

Recent motor control theories suggest that the brain uses internal models to plan and control accurate movements. An internal model is thought to represent how the biomechanics of the arm interacting with the outside world would respond to a motor command; therefore it can be seen as a predictive model of the reafference that helps the system plan ahead. Moreover, adaptation studies show that humans can learn multiple internal models. It is not clear, however, whether and how contextual cues are used to switch among competing internal models, which are required to compensate for altered environments. To investigate this question, we asked healthy participants to perform center-out pointing movements under normal and distorted visual feedback (0 degrees , 30 degrees counterclockwise, and 60 degrees clockwise rotation of hand-screen cursor relationships) conditions. The results suggest that humans can learn multiple environments simultaneously and can use contextual cues to facilitate adaptation and to recall the appropriate internal model of the visuomotor transformation.

Adaptation, Physiological↗

PubMed1996

Mutation in p53 and de-regulation of p53-related gene expression in three human cell lines immortalized with 4-nitroquinoline 1-oxide or 60Co gamma rays.

In vitro models of malignant transformation of human cells may provide considerable insight into the mechanisms of multi-step carcinogenesis. It is well established that normal human cells must be immortalized before they can be malignantly transformed; however, they are stringently destined for aging and are rarely immortalized. The mechanism of cellular aging and immortalization is still unknown. We detected expression of only mutated p53 mRNA by direct sequencing of the reverse-transcribed mRNA in 3 human cell lines immortalized either with 4-nitroquinoline 1-oxide or with 60Co gamma rays. Consequently, only the mutated pS3 protein was expressed in each immortalized cell line. The expression of sdiI/p21 and mdm2, both of which are positively regulated by wild-type p53, was significantly down-regulated in the immortalized cell lines, resulting in over-expression of cdk2 and cdk4. Introduction of the sdiI/p21 gene into these cells was followed by a remarkable decrease in their ability to synthesize DNA. These results indicate that the p53 cascade may play an important role in the immortalization of human cells.

4-Nitroquinoline-1-oxide↗

PubMed1993

Synergistic effects of ionizing radiation and 60 Hz magnetic fields.

Experiments designed to evaluate the synergistic production of clastogenic effects by ionizing radiation and 60 Hz magnetic fields were performed using human lymphocytes from peripheral blood. Following exposure to ionizing radiation, cells were cultured in 60 Hz magnetic fields having field strengths up to 1.4 mT. Cells exposed to both ionizing radiation and 60 Hz magnetic fields demonstrated an enhanced frequency of near tetraploid chromosome complements, a feature not observed following exposure to only ionizing radiation. The results are discussed in the context of a multiple-stage model of cellular transformation, employing both initiating and promoting agents.

Electromagnetic Fields↗

PubMed2004

MHC class I antigen processing pathway defects, ras mutations and disease stage in colorectal carcinoma.

Colorectal tumorigenesis has been associated with the progressive acquisition of a variety of genetic alterations. These include mutations of the Ki-ras proto-oncogene in codons 12 and 13, which account for 85% of genetic changes in colorectal cancer. In murine in vitro models of oncogenic transformation, an association between ras-mediated transformation and downregulation of different components of the MHC class I antigen processing machinery (APM) has been described. In order to investigate whether this association also exists in human tumors, 10 cases of high-grade intraepithelial neoplasia (HIN), as well as primary tumors and autologous lymph node metastases from 42 patients with colorectal carcinoma, were monitored by allele-specific restriction analysis for Ki-ras mutations. In parallel, APM component expression and tumor cell proliferation were analyzed by immunohistochemistry. In comparison to autologous colorectal mucosa, TAP1, LMP2 and tapasin loss was found in 68%, 67% and 80% of HIN, respectively. In contrast, impaired TAP1, LMP2 and tapasin expression was found in 42%, 42% and 63% of primary adenocarcinomas of stage III disease and in 63%, 47% and 79% of the matched lymph node metastases, respectively. More than 60% of colorectal tumor lesions with TAP1, LMP2 and/or tapasin defects displayed Ki-ras mutations. The frequency of TAP1, LMP2 and tapasin loss varied between 33% of primary adenocarcinomas, 40% of HIN to approximately 67% of metastases. These data suggest that i) APM component deficiencies occur more frequently in Ki-ras-mutated colorectal carcinoma lesions and ii) APM abnormalities in conjunction with Ki-ras mutations appear to be associated with disease stage. These findings support the hypothesis that Ki-ras mutations may contribute to immune escape mechanisms of tumors by downregulating the MHC class I APM component expression.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2003

Lactate doublet quantification and lipid signal suppression using a new biexponential decay filter: application to simulated and 1H MRS brain tumor time-domain data.

A new postprocessing filter based on the continuous wavelet transform (CWT) method modeled as a biexponential decay function to isolate the lactate doublet from overlapping lipid resonance(s) and estimate its magnetic resonance spectroscopy (MRS) parameters (signal amplitude, resonance frequencies, and apparent relaxation time (T(*) (2))) is proposed. The new filter employs the same iterative process used in the previously single exponential decay filter. A comparison of the results obtained from application of both filters to simulated data and real (1)H MRS data collected from human blood plasma and brain tumors demonstrates that the new filter provides a better estimate of MRS parameters of lactate, with less computation time. Furthermore, the results show that the new filter is less sensitive to noise and provides a direct estimate of J-coupling value of the lactate doublet.

Adipose Tissue↗

PubMed1989

ras mutations in human leukemia and related disorders.

The clinical association of an increased incidence of acute myelogenous leukemia (AML) with previous chemoradiotherapy, the detection of specific karyotypic changes in these secondary (therapy-induced) cases of AML and the discovery of increasing levels of oncogene-specific RNA in leukemia cells suggest that one potential site of action of environmental agents might be the proto-oncogenes in human hematopoietic stem cells. The location of human proto-oncogenes at the sites of chromosome breaks and/or translocations in cells from some patients with leukemia or lymphoma is a striking observation. These data stimulated research into the mechanism of activation of specific oncogenes that change the biology of human hematopoietic cells. Recent investigations have focused upon several areas that might alter cell biology including: 1) translocation and/or inversion of chromosome fragments containing a proto-oncogene to a location where other gene sequences can stimulate oncogene activation, 2) replication of copy number of proto-oncogenes or increased transcriptional activity and 3) point mutation in proto-oncogenes leading to a structurally altered protein. The third area of research has recently received significant attention with respect to the potential role of three ras genes (c-Harvey-ras, c-Kirsten-ras and N-ras) in human leukemias and myelodysplastic syndromes. Recent studies have proposed a model for leukemogenic transformation of human hematopoietic cells by the product of a mutated ras oncogene. Mutations at codons 12, 13 or 61 of the first exon of its 4.7 Kb of DNA (for c-Ha-ras) have been described. Other data revealing an absence of such mutations in the ras genes of many human leukemias and the absence of detectable transcription of ras genes in many alkylating agent-associated cases of AML, suggest that while ras mutations may be involved in some settings, there are probably multiple genetic pathways to leukemogenic transformation of human hematopoietic cells.

Animals↗

PubMed2002

Calculation and prediction of adsorption excesses on the ternary liquid mixture/air interface from surface tension measurements.

The surface tensions of the n-propanol/ethylene glycol/water ternary mixture and its binary mixtures are measured at 293 K and described by mathematical functions. The experimental data and their descriptions are shown in the three-dimensional space by means of coordinate transformation. A thermodynamic model to calculate and predict ternary adsorption excess data on the ternary liquid mixture/air interface from surface tensions is presented. The calculated adsorption excess data in the ternary triangle are discussed.

Journal Article↗

PubMed2000

Towards a complete description of the structural and dynamic properties of the denatured state of barnase and the role of residual structure in folding.

The detailed characterization of denatured proteins remains elusive due to their mobility and conformational heterogeneity. NMR studies are beginning to provide clues regarding residual structure in the denatured state but the resulting data are too sparse to be transformed into molecular models using conventional techniques. Molecular dynamics simulations can complement NMR by providing detailed structural information for components of the denatured ensemble. Here, we describe three independent 4 ns high-temperature molecular dynamics simulations of barnase in water. The simulated denatured state was conformationally heterogeneous with respect to the conformations populated both within a single simulation and between simulations. Nonetheless, there were some persistent interactions that occurred to varying degrees in all simulations and primarily involved the formation of fluid hydrophobic clusters with participating residues changing over time. The region of the beta(3-4) hairpin contained a particularly high degree of such side-chain interactions but it lacked beta-structure in two of the three denatured ensembles: beta(3-4) was the only portion of the beta-structure to contain significant residual structure in the denatured state. The two principal alpha-helices (alpha1 and alpha2) adopted dynamic helical structure. In addition, there were persistent contacts that pinched off core 2 from the body of the protein. The rest of the protein was unstructured, aside from transient and mostly local side-chain interactions. Overall, the simulated denatured state contains residual structure in the form of dynamic, fluctuating secondary structure in alpha1 and alpha2, as well as fluctuating tertiary contacts in the beta(3-4) region, and between alpha1 and beta(3-4), in agreement with previous NMR studies. Here, we also show that these regions containing residual structure display impaired mobility by both molecular dynamics and NMR relaxation experiments. The residual structure was important in decreasing the conformational states available to the chain and in repairing disrupted regions. For example, tertiary contacts between beta(3-4) and alpha1 assisted in the refolding of alpha1. This contact-assisted helix formation was confirmed in fragment simulations of beta(3-4) and alpha1 alone and complexed, and, as such, alpha1 and beta(3-4) appear to be folding initiation sites. The role of these sites in folding was investigated by working backwards and considering the simulation in reverse, noting that earlier time-points from the simulations provide models of the major intermediate and transition states in quantitative agreement with data from both unfolding and refolding experiments. Both beta(3-4) and alpha1 are dynamic in the denatured state but when they collide and make enough contacts, they provide a loose structural scaffold onto which further beta-strands pack. The beta-structure condenses about beta(3-4), while alpha1 aids in stabilizing beta(3-4) and maintaining its orientation. The resulting beta-structure is relatively planar and loose in the major intermediate. Further packing ensues, and as a result the beta-sheet twists, leading to the major transition state. The structure is still expanded and loops are not well formed at this point. Fine-tuning of the packing interactions and the final condensation of the structure then occurs to yield the native state.

Amino Acid Sequence↗

PubMed1999

Thrombopoietin signal transduction: studies from cell lines and primary cells.

Thrombopoietin (TPO) and its receptor Mpl support all of the developmental step necessary for megakaryocytopoiesis. In the past few years, the signaling pathways utilized by this member of the cytokine receptor family have been extensively studied, especially JAK/STAT, Ras/MAP kinase, Shc, and other adapter molecules. Many if not most of the secondary signaling pathways activated by thrombopoietin have also been identified upon binding of other hematopoietic growth factors to their cognate receptors, making the study of Mpl signaling representative of the field in general. However, identifying unique molecules or combinations of signals that direct megakaryocyte development has been an elusive goal and has led some investigators to conclude that there is little specificity during Mpl signal transduction. In this article we review the data regarding Mpl signaling with particular attention to the methods employed and critical interpretation of the data generated. Future studies will have to focus on primary bone marrow cells and intact animal models rather than transformed cell lines. Furthermore, it is likely that a comprehensive, integrative analysis of the many pathways activated by ligand binding will be necessary to understand the physiology of cytokine signaling.

Animals↗