4,573 research outputs found

    Genetically engineered cardiac pacemaker: stem cells transfected with HCN2 gene and myocytes - a model

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    Artificial biological pacemakers were developed and tested in canine ventricles. Next steps will require obtaining oscillations sensitive to external regulations, and robust with respect to long term drifts of expression levels of pacemaker currents and gap junctions. We introduce mathematical models intended to be used in parallel with the experiments. The models describe human mesenchymal stem cells ({\it hMSC}) transfected with HCN2 genes and connected to myocytes. They are intended to mimic experiments with oscillation induction in a cell pair, in cell culture and in the cardiac tissue. We give examples of oscillations in a cell pair, in a 1 dim cell culture, and oscillation dependence on number of pacemaker channels per cell and number of gap junctions. The models permit to mimic experiments with levels of gene expressions not achieved yet, and to predict if the work to achieve this levels will significantly increase the quality of oscillations. This give arguments for selecting the directions of the experimental work

    Diagnosis of Rosai-Dorfman Disease by Fine Needle Aspiration Cytology in a Case with Cervical Lymphadenopathy and Nasal Mass

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    We report a case of Rosai-Dorfman Disease, a rare non neoplastic proliferative disorder of the cells of macrophage-histiocyte family, in a case with cervical lymphadenopathy and nasal mass diagnosed by fine needle aspiration cytology

    A comparison of haematopoietic stem cells from umbilical cord blood and peripheral blood for platelet production in a microfluidic device

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    Background and objectives: Several sources of haematopoietic stem cells have been used for static culture of megakaryocytes to produce platelets in vitro. This study compares and characterizes platelets produced in shear flow using precursor cells from either umbilical (UCB) or adult peripheral blood (PB). Materials and methods: The efficiency of platelet production of the cultured cells was studied after perfusion in custom-built von Willebrand factor-coated microfluidic flow chambers. Platelet receptor expression and morphology were investigated by flow cytometry and microscopy, respectively. Results: Proliferation of stem cells isolated out of UCB was significantly higher (P < 0 center dot 0001) compared to PB. Differentiation of these cells towards megakaryocytes was significantly lower from PB compared to UCB where the fraction of CD42b/CD41 double positive events was 44 +/- 9% versus 76 +/- 11%, respectively (P < 0 center dot 0001). However, in vitro platelet production under hydrodynamic conditions was more efficient with 7 center dot 4 platelet-like particles per input cell from PB compared to 4 center dot 2 from UCB (P = 0 center dot 02). The percentage of events positive for CD42b, CD41 and CD61 was comparable between both stem cell sources. The mean number of receptors per platelet from UCB and PB was similar to that on blood bank platelets with on average 28 000 CD42b, 57 000 CD61 and 5500 CD49b receptors. Microscopy revealed platelets appearing similar to blood bank platelets in morphology, size and actin cytoskeleton, alongside smaller fragments and source megakaryocytes. Conclusion: This characterization study suggests that platelets produced in vitro under flow either from UCB or from PB share receptor expression and morphology with donor platelets stored in the blood bank

    The MinK Potassium Channel Exists in Functional and Nonfunctional Forms When Expressed in the Plasma Membrane of \u3cem\u3eXenopus\u3c/em\u3e Oocytes

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    The minK protein induces a slowly activating voltage-dependent potassium current when expressed in Xenopus oocytes. In order to measure the levels of minK protein in the plasma membrane, we have modified the minK gene by inserting a 9 amino acid epitope into the N- terminal domain of the protein sequence. When intact live oocytes are injected with the modified minK RNA and subsequently incubated with an antibody to this epitope, specific binding is detected, indicating that the N-terminal domain is extracellular. We found that when oocytes are injected with amounts of minK mRNA up to 50 ng, the levels of protein at the surface are proportional to the amount of injected mRNA. In contrast, the amplitude of the minK current recorded in the oocytes saturates at 1 ng of injected mRNA. Although the amplitude of the currents is not altered by increasing mRNA levels above 1 ng, the kinetics of activation of the current differ in oocytes with high or low levels of minK RNA. In particular, activation is slower with higher levels of minK protein in the plasma membrane. Finally, we find that increasing intracellular cAMP levels, which increases the amplitude of minK currents, does not alter surface expression of the minK protein but produces a small increase in the rate of activation of the current. Our results support a model in which minK protein forms functional potassium channels by association with a factor endogenous to the oocyte

    Sensitivity of normal and malignant human lymphocytes to 5-aminolevulinic acid-mediated photodynamic damage

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    Aim: To compare the sensitivity of normal and malignant human lymphocytes to 5-aminolevulinic acid (ALA) — mediated photodynamic damage. Methods: Blood lymphocytes isolated by Ficoll-sodium metrizoate density gradient from healthy donors (6) and hematologic patients (20) with different forms of lympholeukemia, and also transformed lymphocytes of human B-cell (Raji, Namalwa) and T-cell (MT-4, HUT-78) lines were inestigated. Diagnoses of chronic lymphoproliferative disorders were made on the grounds of morphological, cytochemical and immunocytochemical studies of peripheral blood and bone marrow cells, with immunophenotype determination by monoclonal antibodies to differentiation antigens of T, B lymphocytes and NK cells and immunocytochemical ABC-AP method. Cells of leukemic B- and T-cell lines were cultured in standard RPMI-1640 medium. For photodynamic treatment, the cells were incubated with ALA and then irradiated by a helium-neon laser (wavelength of 633 nm). The number of dead cells was determined in 20 h with trypan blue dye exclusion test. Results: The striking difference in responsiveness to ALA-mediated photodynamic treatment (ALA-PDT) between normal lymphocytes and cells isolated from lymphatic leukemia patients was established. A bulk of leukemic cells (mean for 10 patients with B-CLL — 62.06 ± 4.03%) were destroyed under the lowest ALA-PDT doses tested: 1 mM ALA, irradiation dose of 25 J/cm2. However, it was virtually impossible to attain any appreciable damage of lymphocytes from healthy donors even with the highest treatment doses (5 mM ALA, 150 J/cm2). High sensitivity to ALA-PDT of malignant lymphocytes was confirmed in experiments with human T- and B-cell leukemic cell lines, and in these experiments, an anomalous reaction to the treatment of Raji cells was also detected. The mechanisms of the difference between normal and malignant lymphocytes are discussed in terms of altered heme-synthesis processes in malignant cells. Conclusions: 1) It is shown for the first time that blood lymphocytes from lymphatic leukemia patients are highly sensitive to the damage with ALA-PDT while lymphocytes of normal donors are practically not damaged. 2) Transformed lymphocytes of human T-cell lines are more sensitive than lymphocytes of B-cell lines. 3) Lymphocytes of the Raji line display anomalous dose-effect dependence with ALA-PDT. 4) It is proposed to evaluate the drastic difference in ALA-PDT responsiveness of normal and malignant lymphocytes as a possible simple and low-traumatic test for B-CLL screening among the elderly people.Цель: изучить чувствительность к фотодинамическому воздействию, опосредованному 5-аминолевулиновой кислотой (АЛК), нормальных и малигнизированных лимфоцитов человека. Методы: объектом исследования служили лимфоциты, выделенные с помощью градиента плотности фиколл-верографина из крови здоровых доноров (6) и гематологических больных (20) с различными формами лимфолейкозов, а также трансформированные лимфоциты В-клеточных (Raji, Namalwa) и Т-клеточных (МТ-4, HUT-78) линий человека. Диагноз хронического лимфопролиферативного заболевания ставили на основании морфологического, цитохимического и иммуноцитохимического исследования периферической крови и клеток костного мозга; иммунофенотипирование проводили с использованием панели моноклональных антител к дифференцировочным антигенам Т-, В-лимфоцитов, NK-клеток и АВС-АР-метода. Трансформированные лимфоциты клеточных линий культивировали в стандартной RPMI-1640 среде с 10% эмбриональной телячьей сывороткой. Для фотодинамического воздействия клетки инкубировали с 1,0–5,0 мМ АЛК в течение 4 ч и облучали гелий-неоновым лазером (длина волны – 633 нм), варьируя дозу от 10 до 150 Дж/см2 . Количество погибших клеток после дополнительной 20-часовой инкубации определяли с помощью теста с трипановым синим. Результаты: показано, что нормальные и малигнизированные лимфоциты человека резко отличаются по чувствительности к АЛК-опосредованному фотодинамическому действию (АЛК-ФД). Значительная часть лимфоцитов крови больных лейкозом (в среднем 62,06 ± 4,03% у 10 пациентов с В-клеточным хроническим лимфолейкозом, В-ХЛЛ) погибала при минимальных параметрах воздействия (1 мМ АЛК, доза облучения 25 Дж/см2 ), тогда как даже наивысшая доза (5 мМ АЛК, 150 Дж/см2 ) не влияла на жизнеспособность нормальных лимфоцитов. В опытах in vitro в Т-клеточных линиях отмечали больший процент гибели клеток, чем в В-клеточных. При этом линия Raji отличается парадоксальным отсутствием зависимости количества погибших клеток от дозы воздействия, что, по-видимому, связано с известным для этой линии дефектом сигнальных путей апоптоза. Обсуждаются вероятные механизмы установленных отличий между нормальными и малигнизированными лимфоцитами в контексте альтерации процессов синтеза клетками гема при малигнизации. Выводы: 1) впервые показано, что малигнизированные лимфоциты крови больных лимфолейкозом проявляют высокую чувствительность к АЛК-ФД при фактическом отсутствии такой чувствительности у лимфоцитов здоровых доноров; 2) озлокачествленные лимфоциты Т-клеточных линий более чувствительны к АЛК-ФД по сравнению с В-клеточными; 3) для клеток линии Raji установлена аномальная зависимость доза/эффект; 4) на основе полученных результатов предложен простой и малотравматичный тест для скрининга пожилых людей на наличие В-ХЛЛ

    Strain uses gap junctions to reverse stimulation of osteoblast proliferation by osteocytes

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    Identifying mechanisms by which cells of the osteoblastic lineage communicate in vivo is complicated by the mineralised matrix that encases osteocytes, and thus, vital mechanoadaptive processes used to achieve load‐bearing integrity remain unresolved. We have used the coculture of immunomagnetically purified osteocytes and primary osteoblasts from both embryonic chick long bone and calvariae to examine these mechanisms. We exploited the fact that purified osteocytes are postmitotic to examine both their effect on proliferation of primary osteoblasts and the role of gap junctions in such communication. We found that chick long bone osteocytes significantly increased basal proliferation of primary osteoblasts derived from an identical source (tibiotarsi). Using a gap junction inhibitor, 18β‐glycyrrhetinic acid, we also demonstrated that this osteocyte‐related increase in osteoblast proliferation was not reliant on functional gap junctions. In contrast, osteocytes purified from calvarial bone failed to modify basal proliferation of primary osteoblast, but long bone osteocytes preserved their proproliferative action upon calvarial‐derived primary osteoblasts. We also showed that coincubated purified osteocytes exerted a marked inhibitory action on mechanical strain–related increases in proliferation of primary osteoblasts and that this action was abrogated in the presence of a gap junction inhibitor. These data reveal regulatory differences between purified osteocytes derived from functionally distinct bones and provide evidence for 2 mechanisms by which purified osteocytes communicate with primary osteoblasts to coordinate their activity

    Genomic function during the lampbrush chromosome stage of amphibian oogenesis

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    Throughout its lengthy developmental history the disposition of the genetic material in the amphibian oocyte nucleus differs from that in other cell types. The chromosomes in the oocyte nucleus, arrested for the whole of oogenesis at the prophase of the first meiotic division, are known to contain at least the tetraploid amount of DNA.(1,2) Oogenesis in amphibia requires months or even years to complete, depending on the species

    Controlled Delivery of Sonic Hedgehog Morphogen and Its Potential for Cardiac Repair

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    The morphogen Sonic hedgehog (Shh) holds great promise for repair or regeneration of tissues suffering ischemic injury, however clinical translation is limited by its short half-life in the body. Here, we describe a coacervate delivery system which incorporates Shh, protects it from degradation, and sustains its release for at least 3 weeks. Shh released from the coacervate stimulates cardiac fibroblasts to upregulate the expression of multiple trophic factors including VEGF, SDF-1α, IGF-1, and Shh itself, for at least 48 hours. Shh coacervate also demonstrates cytoprotective effects for cardiomyocytes in a hydrogen peroxide-induced oxidative stress environment. In each of these studies the bioactivity of the Shh coacervate is enhanced compared to free Shh. These results warrant further investigation of the in vivo efficacy of Shh coacervate for cardiac repair. © 2013 Johnson, Wang
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