Ordered Self-Assembly Mechanism of a Spherical Oncoprotein Oligomer Triggered by Zinc Removal and Stabilized by an Intrinsically Disordered Domain

BACKGROUND: Self-assembly is a common theme in proteins of unrelated sequences or functions. The human papillomavirus E7 oncoprotein is an extended dimer with an intrinsically disordered domain, that can form large spherical oligomers. These are the major species in the cytosol of HPV transformed and cancerous cells. E7 binds to a large number of targets, some of which lead to cell transformation. Thus, the assembly process not only is of biological relevance, but represents a model system to investigate a widely distributed mechanism. METHODOLOGY/PRINCIPAL FINDINGS: Using various techniques, we monitored changes in secondary, tertiary and quaternary structure in a time course manner. By applying a robust kinetic model developed by Zlotnik, we determined the slow formation of a monomeric "Z-nucleus" after zinc removal, followed by an elongation phase consisting of sequential second-order events whereby one monomer is added at a time. This elongation process takes place at a strikingly slow overall average rate of one monomer added every 28 seconds at 20 µM protein concentration, strongly suggesting either a rearrangement of the growing complex after binding of each monomer or the existence of a "conformation editing" mechanism through which the monomer binds and releases until the appropriate conformation is adopted. The oligomerization determinant lies within its small 5 kDa C-terminal globular domain and, remarkably, the E7 N-terminal intrinsically disordered domain stabilizes the oligomer, preventing an insoluble amyloid route. CONCLUSION: We described a controlled ordered mechanism with features in common with soluble amyloid precursors, chaperones, and other spherical oligomers, thus sharing determining factors for symmetry, size and shape. In addition, such a controlled and discrete polymerization reaction provides a valuable tool for nanotechnological applications. Finally, its increased immunogenicity related to its supramolecular structure is the basis for the development of a promising therapeutic vaccine candidate for treating HPV cancerous lesions

Mechanical coupling of microtubule-dependent motor teams during peroxisome transport in Drosophila S2 cells

Background: Intracellular transport requires molecular motors that step alongcytoskeletal filaments actively dragging cargoes through the crowded cytoplasm. Here,we explore the interplay of the opposed polarity motors kinesin-1 and cytoplasmicdynein during peroxisome transport along microtubules in Drosophila S2 cells.Methods: We used single particle tracking with nanometer accuracy and millisecondtime resolution to extract quantitative information on the bidirectional motion oforganelles. The transport performance was studied in cells expressing a slow chimericplus-end directed motor or the kinesin heavy chain. We also analyzed the influence ofperoxisomes membrane fluidity in methyl--ciclodextrin treated cells. The experimentaldata was also confronted with numerical simulations of two well-established tug of warscenarios.SCResults and conclusions: The velocity distributions of retrograde and anterogradeperoxisomes showed a multimodal pattern suggesting that multiple motor teams drivetransport in either direction. The chimeric motors interfered with the performance ofanterograde transport and also reduced the speed of the slowest retrograde team. Inaddition, increasing the fluidity of peroxisomes membrane decreased the speed of theslowest anterograde and retrograde teams.General Significance: Our results support the existence of a crosstalk betweenopposed-polarity motor teams. Moreover, the slowest teams seem to mechanicallycommunicate with each other through the membrane to trigger transport.Fil: De Rossi, María Cecilia. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Wetzler, Diana E.. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Benseñor, Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: De Rossi, María Emilia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires; ArgentinaFil: Sued, Raquel Mariela. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Cálculo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rodríguez, Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Cálculo; ArgentinaFil: Gelfand, Vladimir. Northwestern University; Estados UnidosFil: Bruno, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires; ArgentinaFil: Levi, Valeria. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

BCR-ABL1 tyrosine kinase sustained MECOM expression in chronic myeloid leukaemia

MECOM oncogene expression correlates with chronic myeloid leukaemia (CML) progression. Here we show that the knockdown of MECOM (E) and MECOM (ME) isoforms reduces cell division at low cell density, inhibits colony-forming cells by 34% and moderately reduces BCR-ABL1 mRNA and protein expression but not tyrosine kinase catalytic activity in K562 cells. We also show that both E and ME are expressed in CD34<sup>+</sup> selected cells of both CML chronic phase (CML-CP), and non-CML (normal) origin. Furthermore, MECOM mRNA and protein expression were repressed by imatinib mesylate treatment of CML-CP CD34<sup>+</sup> cells, K562 and KY01 cell lines whereas imatinib had no effect in non-CML BCR-ABL1 −ve CD34<sup>+</sup> cells. Together these results suggest that BCR-ABL1 tyrosine kinase catalytic activity regulates MECOM gene expression in CML-CP progenitor cells and that the BCR-ABL1 oncoprotein partially mediates its biological activity through MECOM. MECOM gene expression in CML-CP progenitor cells would provide an in vivo selective advantage, contributing to CML pathogenesis

Retear of anterior cruciate ligament grafts in female basketball players: a case series

<p>Abstract</p> <p>Background</p> <p>Incidence of anterior cruciate ligament (ACL) injuries in young female basketball players is higher than that in male basketball players. Graft retears are more frequent with the increasing number of ACL reconstructions. The present study aimed to examine the incidence of retears in competitive female basketball players.</p> <p>Methods</p> <p>Sixty-four female basketball players (aged 12 to 29 years) who underwent primary anatomic double-bundle ACL reconstruction using hamstring grafts participated in the study. We investigated incidence, mechanism, and patient characteristics of ACL graft retears. Mann-Whitney <it>U </it>test was used for statistical analysis, and the level of significance was determined at <it>P </it>< 0.05.</p> <p>Results</p> <p>Six patients suffered from ACL graft retear (9.4%). Mean duration between primary ACL reconstruction and incidence of retears was 11.7 months. However, there were no other postoperative graft ruptures after 24 months. Primary injury and retear mechanisms varied by patient. At six months after the primary ACL reconstruction surgery, mean quadriceps and hamstring strengths were 81% and 87%, respectively, indicating favorable recovery of muscle strength. However, preoperative quadriceps and hamstring strength in the retear group were 65% and 71%, respectively. In particular, preoperative quadriceps strength in the retear group demonstrated a lower value than that in the uninjured group (<it>P </it>< 0.05).</p> <p>Conclusions</p> <p>We observed a high incidence of ACL graft retears in competitive female basketball players, as previously reported. Considering the timing of graft retear occurrences, an early return to playing basketball should be avoided following ACL reconstruction. Closer attention should be paid to player preoperative condition, as well as muscle strength and postoperative status.</p

New Human Papilloma Virus E2 Transcription Factor Mimics: A Tripyrrole-Peptide Conjugate with Tight and Specific DNA-Recognition

BACKGROUND: Human papillomavirus (HPV) is the main causative agent of cervical cancer, particularly high risk strains such us HPV-16, -18 and -31. The viral encoded E2 protein acts as a transcriptional modulator and exerts a key role in viral DNA replication. Thus, E2 constitutes an attractive target for developing antiviral agents. E2 is a homodimeric protein that interacts with the DNA target through an α-helix of each monomer. However, a peptide corresponding to the DNA recognition helix of HPV-16 E2 binds DNA with lower affinity than its full-length DNA binding domain. Therefore, in an attempt to promote the DNA binding of the isolated peptide, we have designed a conjugate compound of the E2 α-helix peptide and a derivative of the antibiotic distamycin, which involves simultaneous minor- and major-groove interactions. METHODOLOGY/PRINCIPAL FINDINGS: An E2 α-helix peptide-distamycin conjugate was designed and synthesized. It was characterized by NMR and CD spectroscopy, and its DNA binding properties were investigated by CD, DNA melting and gel shift experiments. The coupling of E2 peptide with distamycin does not affect its structural properties. The conjugate improves significantly the affinity of the peptide for specific DNA. In addition, stoichiometric amounts of specific DNA increase meaningfully the helical population of the peptide. The conjugate enhances the DNA binding constant 50-fold, maintaining its specificity. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that peptide-distamycin conjugates are a promising tool to obtain compounds that bind the E2 target DNA-sequences with remarkable affinity and suggest that a bipartite major/minor groove binding scaffold can be a useful approach for therapeutic treatment of HPV infection

Histone H4 acetylation by immunohistochemistry and prognosis in newly diagnosed adult acute lymphoblastic leukemia (ALL) patients

Background: Histone deacetylase (HDAC) inhibitors are a novel anti-tumor therapy. To determine whether HDAC inhibitors may be useful in the treatment of adult acute lymphoblastic leukemia (ALL), we examined the acetylation of histone H4 by immunohistochemistry in newly diagnosed ALL patients and evaluated the impact of acetylation on complete remission (CR) rate, relapse-free survival (RFS), and overall survival (OS). Methods: Patients >= 18 years of age and an available diagnostic bone marrow biopsy were evaluated. Cox proportional hazards analysis was used to identify univariate and multivariate correlates of CR, RFS, and OS. The variables histone H4 acetylation (positive or negative), white blood count, cytogenetic (CG) risk group (CALGB criteria), and age were used in multivariate analysis. Results: On multivariate analysis, histone acetylation was associated with a trend towards an improved OS (for all CG risk groups) (HR = 0.51, p = 0.09). In patients without poor risk CG, there was an impressive association between the presence of histone acetylation and an improved CR rate (OR 3.43, p = 0.035), RFS (HR 0.07, p = 0.005), and OS (HR 0.24, p = 0.007). This association remained statistically significant in multivariate analysis. Conclusions: These data provide a rationale for the design of novel regimens incorporating HDAC inhibitors in ALL

Azimuthal anisotropy and correlations in p+p, d+Au and Au+Au collisions at 200 GeV

We present the first measurement of directed flow ($v_1$) at RHIC. $v_1$ is found to be consistent with zero at pseudorapidities $\eta$ from -1.2 to 1.2, then rises to the level of a couple of percent over the range $2.4 < |\eta| < 4$. The latter observation is similar to data from NA49 if the SPS rapidities are shifted by the difference in beam rapidity between RHIC and SPS. Back-to-back jets emitted out-of-plane are found to be suppressed more if compared to those emitted in-plane, which is consistent with {\it jet quenching}. Using the scalar product method, we systematically compared azimuthal correlations from p+p, d+Au and Au+Au collisions. Flow and non-flow from these three different collision systems are discussed.Comment: Quark Matter 2004 proceeding, 4 pages, 3 figure

Azimuthal anisotropy: the higher harmonics

We report the first observations of the fourth harmonic (v_4) in the azimuthal distribution of particles at RHIC. The measurement was done taking advantage of the large elliptic flow generated at RHIC. The integrated v_4 is about a factor of 10 smaller than v_2. For the sixth (v_6) and eighth (v_8) harmonics upper limits on the magnitudes are reported.Comment: 4 pages, 6 figures, contribution to the Quark Matter 2004 proceeding

Partonic flow and ϕ\phi-meson production in Au+Au collisions at sNN\sqrt{s_{NN}} = 200 GeV

We present first measurements of the $\phi$-meson elliptic flow ($v_{2}(p_{T})$) and high statistics $p_{T}$ distributions for different centralities from $\sqrt{s_{NN}}$ = 200 GeV Au+Au collisions at RHIC. In minimum bias collisions the $v_{2}$ of the $\phi$ meson is consistent with the trend observed for mesons. The ratio of the yields of the $\Omega$ to those of the $\phi$ as a function of transverse momentum is consistent with a model based on the recombination of thermal $s$ quarks up to $p_{T}\sim 4$ GeV/$c$, but disagrees at higher momenta. The nuclear modification factor ($R_{CP}$) of $\phi$ follows the trend observed in the $K^{0}_{S}$ mesons rather than in $\Lambda$ baryons, supporting baryon-meson scaling. Since $\phi$-mesons are made via coalescence of seemingly thermalized $s$ quarks in central Au+Au collisions, the observations imply hot and dense matter with partonic collectivity has been formed at RHIC.Comment: 6 pages, 4 figures, submit to PR

Strange Resonance Production in p+p and Au+Au Collisions at RHIC Energies

Resonance yields and spectra from elementary p+p and Au+Au collisions at $\sqrt{s_{\rm NN}} =$ 200 GeV from the STAR experiment at RHIC are presented and discussed in terms of chemical and thermal freeze-out conditions. Thermal models do not adequately describe the yields of the resonance production in central Au+Au collisions. The approach to include elastic hadronic interactions between chemical freeze-out and thermal freeze-out suggests a time of $\Delta \tau>$5 fm/c.Comment: 4 pages, 7 figures, proceedings of the Quark Matter 2004, in Oakland, California, to be published in Journal of Physics G: Nuclear and Particle Physic