Marcadores virologicos no convencionales en pacientes infectados con el virus de la inmunodeficiencia humana: ADN HIV-T, ADN HIV-2LTR y ARN de HIV / Non conventional virological markers in HIV-infected patients: T-HIV DNA, 2LTR-HIV DNA and HIV RNA

La terapia antirretrovial de alta eficacia (TAAE) induce una reducción marcada y persisatente de la viremia plasmática, contribuvendo a disminuir la mortalidad de los pacientes HIV-positivos. Así, la carga viral (CV) es el método de referencia para evaluar la eficacia terapéutica. Sin embargo, aun en presencia de una TAAE eficiente no se ha logrado la erradicación viral. En este estudio analizamos la presencia del ADN total de HIV (ADN HIV-T), del ADN no integrado con 2LTR (ADN HIV-2LTR) y del ARN de HIV, en un grupo de 55 pacientes HIV-positivos en distintos estadios clínicos, con y sin TAAE, mediante ensayos de PCR con revelado colorimétrico en microplaca, optimizados en nuestro laboratorio. La sensibilidad clínica de ARN del HIV fue evaluada con el bDNA, resultando del 74% y del 64%, respectivamente, con una concordancia del 85%. Este ensayo podría utilizado en el seguimiento de pacientes bajo TAAE. EI ADN HIV-2LTR resultó positivo en el 54% aunque estuvo ausente en pacientes con elevada CV. Este marcador se considereba un producto lábil y su presencia se asociaba a infección reciente. Sin embargo, actuales evidencias ponen en discusión su estabilidad por lo que su significado clínico debe ser reconsiderado. La ausencia del ADN HIV-2LTR en pacientes con CV detectable puede relacionarse con la heterogeneidade de la secuencia utilizada para su detección. EI ADN HIV-T estuvo presente en el 100% de las muestras y resultaría relevante como marcador de remisión cuando se dispongan de terapias que efectivamente erradiquen la infección. (AU)

Inhibition of focal adhesion kinase by antisense oligonucleotides enhances the sensitivity of breast cancer cells to camptothecins

This study shows a strong association between cell attachment to substratum and activation of beta 1-integrin-signaling with resistance to the camptothecin derivative topotecan (TPT) in breast cancer cells. We propose a mechanistic-driven approach to sensitize the cells to camptothecins. ZR-75-1 anchorage-dependent breast cancer cell line, its derivative 9D3S suspension cells (9D3S-S), and 9D3S cells attached to fibronectin-coated plates (9D3S-A) were treated with TPT (1 microM) or CPT-11 (40 microM) for 48 h. Programmed cell death (PCD), as shown by poly(ADP-ribose) polymerase (PARP), pro-caspase-3 and pro-caspase-9 cleavage, was observed in 9D3S-S cells but not in ZR-75-1 or 9D3S-A cells. Because p125 focal adhesion kinase (FAK) is a transducer in the beta 1-integrin signaling pathway, it is essential to cell adhesion and it is overexpressed in metastatic breast cancer, we hypothesized that attenuation of FAK might enhance the sensitivity of breast cancer cells to camptothecins. Moreover, inhibition of FAK gene expression by a phosphorothioated antisense oligodeoxynucleotide targeting the portion of the gene encoding amino acids 262-268, increased the sensitivity of ZR-75-1, MDA-MB-231 and MCF7 breast cancer cells to treatment with TPT or CPT-11. (AU)

Correlative microscopy of cerebellar Golgi cells

The cerebellar Golgi cells of mouse, teleost fish, primate and human species have been studied by means of light and Golgi light microscopic techniques, confocal laser scanning microscopy, slicing technique, ethanol-cryofracturing and freeze-fracture methods for scanning electron microscopy and ultrathin sectioning and freeze-etching replicas for transmission electron microscopy. The Golgi cells appeared in the granular layer as polygonal, stellate, round or fusiform macroneurons surrounded by the granule cell groups. They exhibited ascending dendrites toward the molecular layer and horizontal dendrites and a short beaded axonal plexus confined to the granular layer. Scanning electron microscopy revealed their three-dimensional neuronal geometry and smooth outer surfaces. Freeze-fracture method for SEM showed the stereospatial cytoplasmic arrangement of endoplasmic reticulum, cell organelles and nuclear envelope. By means of transmission electron microscopy the asymmetric synaptic connections of Golgi cell horizontal dendrites--with mossy fiber rosettes at the cerebellar glomerulus--and of Golgi cell axons--with granule cell dendrites at the periphery of glomerular region--were identified. At the molecular layer, Golgi cell ascending dendrites exhibited short neckless spines establishing asymmetric contacts with granule cell axons or parallel fibers. Shaft asymmetric axodendritic and axospinodendritic contacts between Golgi cell dendrites and climbing fibers were also found in the molecular layer.(AU)

The role of plasminogen activator receptor in cancer invasion and dormancy

Urokinase plasminogen activator receptor (uPAR) has been identified some 15 years ago and the anticipation was that is presence on the cell surface will provide a focus for anchoring uPA and possibly protect the enzyme from native inhibitors. The studies of the last decade have shown that uPA localized to the surface of cells by uPAR is indeed an important factor in the process of cancer cell invasion and metastasis. We developed a chick embryo model in which we showed that uPAR is crucial in invasion of stroma and in intravasation (breaching of the blood vessels walls). More recently and unexpectedly, uPAR-a protein anchored in the outer leaflet of the plasma membrane, has been shown to initiate signal transduction events and affect cell migration. We have shown that uPAR co-associates with fibronectin binding integrin, alpha5beta1, activates them and that this interaction leads to a greatly increased level of active ERK. When the association between uPAR and integrin or integrin and fibronectin are interrupted either by reduction of surface uPAR expression, or by other means, human carcinoma cells enter a state of protracted dormancy. We show that very high levels of active ERK are required to keep cancer cells proliferating in vivo. (AU)

Differentiation and breast cancer

The mammary gland is an organ whose size, shape and function undergo fundamental changes during the various phases of a womans growth. Although the development of the mammary gland begins during infancy, the most dramatic changes occur with the initiation of puberty. Pregnancy and lactation complete the functional development of the organ, which regresses during menopause. Epidemiological and experimental studies have demonstrated that certain hormonal influences, especially those related to reproduction, modify the risk of developing breast cancer. Thus, a full term pregnancy completed before the age of 24 years significantly reduces the lifetime incidence of breast cancer. Although the mechanism through which pregnancy protects the breast from breast cancer has not been clearly established, experimental models of mammary carcinogenesis have allowed researchers to determine that pregnancy inhibits the initiation of the neoplastic process through the induction of a complete differentiation of the mammary gland. This process activates specific genes, which in turn modify the response of the organ to ulterior hormonal changes. It is postulated that the same mechanism might be responsible for the protective effect of a womans early first full term pregnancy. The greater incidence of breast cancer observed in nulliparous women correlates well with the greater susceptibility of the virgin rat to develop mammary carcinomas when exposed to chemical carcinogens. The successful induction of malignant transformation in the virgin animal mammary epithelium is due to the presence of undifferentiated structures with a high rate of cell proliferation. These structures are eliminated by pregnancy. The breast of nulliparous women retains those undifferentiated structures, which increase the predisposition of the organ to undergo malignant transformation, which will manifest itself clinically several years after its initiation. The correlation of human epidemiological, clinical and experimental data with those data obtained in rodent experimental models lends support to this hypothesis.(Au)

Vesicular transport: implications for cell polarity

In polarized cells intracellular sorting of plasma membrane proteins occurs to a large extent at the trans-Golgi network, giving rise to vesicles destined for distinct plasma membrane domains. This review discusses the several pathways, both direct and indirect, which lead to protein incorporation into the correct cell surface, as well as the mechanisms involved. Proteins contain signals which direct their incorporation into the distinct vesicles destined for plasma membrane microdomains. Specific coat proteins are involved in vesicle assembly and are likely to play a role in the generation of discrete vesicle populations. Molecules involved in vesicle docking and fusion may also add specificity to the targeting process.(AU)

Mechanism of formation of post Golgi vesicles from TGN membranes: Arf-dependent coat assembly and PKC-regulated vesicle scission

We have developed an experimental system that utilizes purified Golgi fractions obtained from virus infected infected MDCK cells to reproduce in vitro the process of vesicle generation in the trans Golgi network, an important site for the sorting of proteins addressed to the plasma membrane, secretory vesicles, or lysosomes. Using an integrated biochemical and electron microscopic approach, we have shown that the formation of post Golgi vesicles carrying proteins destined to both plasma membrane domains of epithelial cells requires the activation of an ArF-like GTP-binding protein that serves to promote the assembly of the protein coat necessary to deform the donor membrane and generate a vesicle. The formation of the post Golgi vesicles also requires the participation of a Golgi membrane-associated Protein Kinase C, but not its phosphorylating activity. Other authors have shown that this is also the case for the PKC activation of the enzyme phospholipase D, which generates phosphatidic acid from phosphatidyl choline and may be involved in remodeling of membranes. We have been able to dissect the process of post Golgi vesicle generation into two sequential stages, one of coat assembly and bud formation, and a subsequent one of vesicle scission. The first stage can occur at 20 degrees C and requires the activation of the Arf protein necessary for coat assembly. The second stage does not require nucleotides or an energy supply, but requires cytosolic proteins, and in particular, an NEM sensitive membrane scission promoting activity that operates only at a higher temperature of incubation. Because various PKC inhibitors blocked vesicle scission without preventing bud formation, we propose that the PKC is required for the activation of a PLD in the TGN, which leads to remodeling of the donor membrane and the severing of connections between the emerging vesicles and the membranes.(AU)

Coupling between charge movement and pore opening in voltage dependent potassium channels

Ionic and gating currents from Shaker K+ channels were characterized with the cut-open oocyte voltage clamp (COVG) technique. Experiments were performed in normally conducting channels and in channels with the W434F mutation which completely abolished ion conduction without affecting the voltage dependence of gating charge. Subtracted and unsubtracted gating currents with the COVG technique, and gating currents recorded in cell attached macro-patches had the same properties and time course. However, OFF gating currents and ionic deactivation tails became slower after patch excision. Gating currents had the following salient properties: 1) the turn-on of the gating current shows a rising phase, 2) the more negative position of the charge-voltage curve (Q-V) vs. the conductance-voltage (G-V) curve and the charge displacement by hyperpolarizing prepulses indicate that a large fraction of the voltage-dependence occurs in the transitions between closed states, 3) the Q-V relationship showed two component with different half activation potential and effective valence; the one appearing at more negative potential had a shallower voltage dependence, while the one at more depolarized potentials had a larger effective valence and correlated with channel opening, 4) ionic and gating currents were similarly time shifted by preceding hyperpolarizing and depolarizing pulses which substantiates the relationship between charge movement and channel opening, 5) in the wild type Shaker K+ clone with fast inactivation, the OFF gating charge is partially immobilized for large depolarizing pulses, while in the mutant channel lacking inactivation the charge is recovered quickly at the end of the pulse, indicating that the channel blockade by the inactivating particle slows down charge recovery, 6) the OFF gating current rapidly returns for small depolarizations, while for larger pulses which open the channel the OFF gating current return is delayed suggesting that the closed to open transitions carry little charge and 7) in the W434F mutant with the conserved amino terminus large depolarizations that would have opened the channel induced OFF charge immobilization, indicating that although the conduction pathway was not functional, the channel can still undergo the closed-open conformation in response to voltage changes. In conclusion, the kinetic properties of gating currents discard equal and independent gating subunits with two positions. (AU)

Modifications of antitumor defenses by tumor derived factors and by superantigens

There is ample evidence that tumor development can be affected by the interactions between the growing neoplasms and the immune system. The balance of these interactions is tilted in favor of tumor growth in many cases due to the production of cytokines and other factors by the tumor cells. These factors can modulate the immune system either by direct interactions with immune cells or by indirect means, which include downregulation of the synthesis of other cytokines or products necessary for the activity of a given effector cell. In addition, endogenous retroviral superantigens, with their capacity of eliminating part of the T cell repertoire and possibly by other effects on cells of the T and B cell lineages, may provide the tumor cells an escape from the otherwise efficient antitumor host defenses (Au)