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At least 91 records · Page 5

Cameleon calcium indicator reports cytoplasmic calcium dynamics in Arabidopsis guard cells

Cytoplasmic free calcium ([Ca2+]cyt) acts as a stimulus-induced second messenger in plant cells and multiple signal transduction pathways regulate [Ca2+]cyt in stomatal guard cells. Measuring [Ca2+]cyt in guard cells has previously required loading of calcium-sensitive dyes using invasive and technically difficult micro-injection techniques. To circumvent these problems, we have constitutively expressed the pH-independent, green fluorescent protein-based calcium indicator yellow cameleon 2.1 in Arabidopsis thaliana (Miyawaki et al. 1999; Proc. Natl. Acad. Sci. USA 96, 2135-2140). This yellow cameleon calcium indicator was expressed in guard cells and accumulated predominantly in the cytoplasm. Fluorescence ratio imaging of yellow cameleon 2.1 allowed time-dependent measurements of [Ca2+]cyt in Arabidopsis guard cells. Application of extracellular calcium or the hormone abscisic acid (ABA) induced repetitive [Ca2+]cyt transients in guard cells. [Ca2+]cyt changes could be semi-quantitatively determined following correction of the calibration procedure for chloroplast autofluorescence. Extracellular calcium induced repetitive [Ca2+]cyt transients with peak values of up to approximately 1.5 microM, whereas ABA-induced [Ca2+]cyt transients had peak values up to approximately 0.6 microM. These values are similar to stimulus-induced [Ca2+]cyt changes previously reported in plant cells using ratiometric dyes or aequorin. In some guard cells perfused with low extracellular KCl concentrations, spontaneous calcium transients were observed. As yellow cameleon 2.1 was expressed in all guard cells, [Ca2+]cyt was measured independently in the two guard cells of single stomates for the first time. ABA-induced, calcium-induced or spontaneous [Ca2+]cyt increases were not necessarily synchronized in the two guard cells. Overall, these data demonstrate that that GFP-based cameleon calcium indicators are suitable to measure [Ca2+]cyt changes in guard cells and enable the pattern of [Ca2+]cyt dynamics to be measured with a high level of reproducibility in Arabidopsis cells. This technical advance in combination with cell biological and molecular genetic approaches will become an invaluable tool in the dissection of plant cell signal transduction pathways.

Non-NASA Center↗

Hypergravity signal transduction in HeLa cells with concomitant phosphorylation of proteins immunoprecipitated with anti-microtubule-associated protein antibodies

It is shown that hypergravity (35g) stimulates the production of inositol 1,4,5-trisphosphate (IP3) and decreases adenosine 3-prime,5-prime-cyclic monophosphate (cAMP) levels in HeLa cells. It is proposed that IP3 and cAMP may act as second messengers in hypergravity signal transduction. Phosphorylation of microtubule-associated proteins in both the detergent-soluble and -insoluble fractions suggests that cytoskeletal structures may be influenced by gravity.

Kumei, Yasuhiro↗

Inhibition of MALT1 and BCL2 Induces Synergistic Antitumor Activity in Models of B-Cell Lymphoma

The activated B cell (ABC) subset of diffuse large B-cell lymphoma (DLBCL) is characterized by chronic B-cell receptor signaling and associated with poor outcomes when treated with standard therapy. In ABC-DLBCL, MALT1 is a core enzyme that is constitutively activated by stimulation of the B-cell receptor or gain-of-function mutations in upstream components of the signaling pathway, making it an attractive therapeutic target. We discovered a novel small-molecule inhibitor, ABBV-MALT1, that potently shuts down B-cell signaling selectively in ABC-DLBCL preclinical models leading to potent cell growth and xenograft inhibition. We also identified a rational combination partner for ABBV-MALT1 in the BCL2 inhibitor, venetoclax, which when combined significantly synergizes to elicit deep and durable responses in preclinical models. This work highlights the potential of ABBV-MALT1 monotherapy and combination with venetoclax as effective treatment options for patients with ABC-DLBCL.

59 BASIC BIOLOGICAL SCIENCES↗

Parathyroid hormone-dependent signaling pathways regulating genes in bone cells

Parathyroid hormone (PTH) is an 84-amino-acid polypeptide hormone functioning as a major mediator of bone remodeling and as an essential regulator of calcium homeostasis. PTH and PTH-related protein (PTHrP) indirectly activate osteoclasts resulting in increased bone resorption. During this process, PTH changes the phenotype of the osteoblast from a cell involved in bone formation to one directing bone resorption. In addition to these catabolic effects, PTH has been demonstrated to be an anabolic factor in skeletal tissue and in vitro. As a result, PTH has potential medical application to the treatment of osteoporosis, since intermittent administration of PTH stimulates bone formation. Activation of osteoblasts by PTH results in expression of genes important for the degradation of the extracellular matrix, production of growth factors, and stimulation and recruitment of osteoclasts. The ability of PTH to drive changes in gene expression is dependent upon activation of transcription factors such as the activator protein-1 family, RUNX2, and cAMP response element binding protein (CREB). Much of the regulation of these processes by PTH is protein kinase A (PKA)-dependent. However, while PKA is linked to many of the changes in gene expression directed by PTH, PKA activation has been shown to inhibit mitogen-activated protein kinase (MAPK) and proliferation of osteoblasts. It is now known that stimulation of MAPK and proliferation by PTH at low concentrations is protein kinase C (PKC)-dependent in both osteoblastic and kidney cells. Furthermore, PTH has been demonstrated to regulate components of the cell cycle. However, whether this regulation requires PKC and/or extracellular signal-regulated kinases or whether PTH is able to stimulate other components of the cell cycle is unknown. It is possible that stimulation of this signaling pathway by PTH mediates a unique pattern of gene expression resulting in proliferation in osteoblastic and kidney cells; however, specific examples of this are still unknown. This review will focus on what is known about PTH-mediated cell signaling, and discuss the established or putative PTH-regulated pattern of gene expression in osteoblastic cells following treatment with catabolic (high) or anabolic (low) concentrations of the hormone.

NASA Discipline Musculoskeletal↗

Nondestructive evaluation techniques for nickel-cadmium aerospace battery cells

The ac impedance characteristics of Ni-Cd cells as an in-situ, nondestructive means of determining cell lifetime, particularly with respect to the probability of premature failure were evaluated. Emphasis was on evaluating Ni-Cd cell impedance over a wide frequency range (10,000 to 0.0004 Hz) as the cells were subjected to charge/discharge cycle testing. The results indicate that cell degradation is reflected in the low frequency (Warburg) impedance characteristics associated with diffusion processes. The Warburg slope (W) was found to steadily increase as a function of cell aging for completely discharged cells. In addition, based on data for two cells, a high or rapidly increasing value for W signals imminent cell failure by one mechanism. Degradation by another mechanism is apparently reflected in a fall-off (roll-over) of W at lower frequencies. As a secondary result, the frequency dependence of the absolute cell impedance at low frequencies (5 - 500 mHz) was found to be a good indication of the cell state-of-charge.

Haak, R.↗

Chloroplast Stress Signals: Control of Retrograde Signaling, Chloroplast Turn-Over, and Cell Fate Decisions

Chloroplasts (photosynthetic plastids) are semiautonomous organelles that contain their own small genomes. The proteomes of chloroplasts, however, are a mixture of plastid and nuclear-encoded proteins. Chloroplasts perform photosynthesis, which is prone to damaging the organelles, leading to the production of reactive oxygen species (ROS) that damage the cell under environmental stresses. Thus, for the cell to maintain proper chloroplast function, efficient photosynthesis, and avoid ROS damage, it relies on complex crosstalk between the chloroplast, the nucleus, other organelles within the cell, and the cytoplasm in between. This communication involves retrograde signals from chloroplasts to control nuclear gene expression, programmed cell death (PCD), and chloroplast degradation. Here we review these signals with an emphasis on the roles of the ROS singlet oxygen ( 1 O 2 ) and plastid gene expression. We cover (1) recent work on understanding how multiple 1 O 2 signaling pathways can be initiated within stressed chloroplasts, (2) how individualized post-translational regulatory systems allow chloroplasts to control their proteomes and degradation, and (3) how chloroplast signals ultimately control cell fate decisions, such as PCD, senescence, and vacuole-mediated degradation of chloroplasts (chloroplast quality control). Overall, this chapter discusses how chloroplasts can act as environmental sensors for the cell and allow plants to acclimate to stress and thrive in dynamic environments.

59 BASIC BIOLOGICAL SCIENCES↗

Chronic shear induces caveolae formation and alters ERK and Akt responses in endothelial cells

Caveolae are plasmalemmal domains enriched with cholesterol, caveolins, and signaling molecules. Endothelial cells in vivo are continuously exposed to shear conditions, and their caveolae density and location may be different from that of static cultured cells. Here, we show that chronic shear exposure regulates formation and localization of caveolae and caveolin-1 in bovine aortic endothelial cells (BAEC). Chronic exposure (1 or 3 days) of BAEC to laminar shear increased the total number of caveolae by 45-48% above static control. This increase was due to a rise in the luminal caveolae density without changing abluminal caveolae numbers or increasing caveolin-1 mRNA and protein levels. Whereas some caveolin-1 was found in the plasma membrane in static-cultured cells, it was predominantly localized in the Golgi. In contrast, chronic shear-exposed cells showed intense caveolin-1 staining in the luminal plasma membrane with minimum Golgi association. The preferential luminal localization of caveolae may play an important role in endothelial mechanosensing. Indeed, we found that chronic shear exposure (preconditioning) altered activation patterns of two well-known shear-sensitive signaling molecules (ERK and Akt) in response to a step increase in shear stress. ERK activation was blunted in shear preconditioned cells, whereas the Akt response was accelerated. These results suggest that chronic shear stimulates caveolae formation by translocating caveolin-1 from the Golgi to the luminal plasma membrane and alters cell signaling responses.

NASA Discipline Cell Biology↗

Three-dimensional co-culture process

The present invention relates to a 3-dimensional co-culture process, more particularly to methods or co-culturing at least two types of cells in a culture environment, either in space or in unit gravity, with minimum shear stress, freedom for 3-dimensional spatial orientation of the suspended particles and localization of particles with differing or similar sedimentation properties in a similar spatial region to form 3-dimensional tissue-like structures. Several examples of multicellular 3-dimensional experiences are included. The protocol and procedure are also set forth. The process allows simultaneous culture of multiple cell types and supporting substrates in a manner which does not disrupt the 3-dimensional spatial orientation of these components. The co-cultured cells cause a mutual induction effect which mimics the natural hormonal signals and cell interactions found in the intact organism. This causes the tissues to differentiate and form higher 3-dimensional structures such as glands, junctional complexes polypoid geometries, and microvilli which represent the corresponding in-vitro structures to a greater degree than when the cell types are cultured individually or by conventional processes. This process was clearly demonstrated for the case of two epithelial derived colon cancer lines, each co-cultured with normal human fibroblasts and with microcarrier bead substrates. The results clearly demonstrate increased 3-dimensional tissue-like structure and biochemical evidence of an increased differentiation state. With the present invention a variety of cells may be co-cultured to produce tissue which has 3-dimensionality and has some of the characteristics of in-vitro tissue. The process provides enhanced 3-dimensional tissue which create a multicellular organoid differentiation model.

Wolf, David A.↗

Calcium signals and calcium channels in osteoblastic cells

Calcium (Ca2+) channels are present in non-excitable as well as in excitable cells. In bone cells of the osteoblast lineage, Ca2+ channels play fundamental roles in cellular responses to external stimuli including both mechanical forces and hormonal signals. They are also proposed to modulate paracrine signaling between bone-forming osteoblasts and bone-resorbing osteoclasts at local sites of bone remodeling. Calcium signals are characterized by transient increases in intracellular Ca2+ levels that are associated with activation of intracellular signaling pathways that control cell behavior and phenotype, including patterns of gene expression. Development of Ca2+ signals is a tightly regulated cellular process that involves the concerted actions of plasma membrane and intracellular Ca2+ channels, along with Ca2+ pumps and exchangers. This review summarizes the current state of knowledge concerning the structure, function, and role of Ca2+ channels and Ca2+ signals in bone cells, focusing on the osteoblast.

Non-NASA Center↗

Electrical signal transmission in a bone cell network: the influence of a discrete gap junction

A refined electrical cable model is formulated to investigate the role of a discrete gap junction in the intracellular transmission of electrical signals in an electrically coupled system of osteocytes and osteoblasts in an osteon. The model also examines the influence of the ratio q between the membrane's electrical time constant and the characteristic time of pore fluid pressure, the circular, cylindrical geometry of the osteon, and key simplifying assumptions in our earlier continuous cable model (see Zhang, D., S. C. Cowin, and S. Weinbaum. Electrical signal transmission and gap junction regulation in a bone cell network: A cable model for an osteon. Ann. Biomed. Eng. 25:379-396, 1997). Using this refined model, it is shown that (1) the intracellular potential amplitude at the osteoblastic end of the osteonal cable retains the character of a combination of a low-pass and a high-pass filter as the corner frequency varies in the physiological range; (2) the presence of a discrete gap junction near a resting osteoblast can lead to significant modulation of the intracellular potential and current in the osteoblast for measured values of the gap junction coupling strength; and (3) the circular, cylindrical geometry of the osteon is well simulated by the beam analogy used in Zhang et al.

Non-NASA Center↗

Laser microsurgery of higher plant cell walls permits patch-clamp access

Plasma membranes of guard cells in epidermal peels of Vicia faba and Commelina communis can be made accessible to a patch-clamp pipet by removing a small portion (1-3 micrometers in diameter) of the guard cell wall using a microbeam of ultraviolet light generated by a nitrogen laser. Using this laser microsurgical technique, we have measured channel activity across plasma membranes of V. faba guard cells in both cell-attached and isolated patch configurations. Measurements made in the inside-out patch configuration revealed two distinct K(+)-selective channels. Major advantages of the laser microsurgical technique include the avoidance of enzymatic protoplast isolation, the ability to study cell types that have been difficult to isolate as protoplasts or for which enzymatic isolation protocols result in protoplasts not amenable to patch-clamp studies, the maintenance of positional information in single-channel measurements, reduced disruption of cell-wall-mediated signaling pathways, and the ability to investigate intercellular signaling through studies of cells remaining situated within tissue.

Non-NASA Center↗

Comparison of Standard and Heart-pacer Type 3rd Electrodes in Design Variable Cells

Nine packs of sealed aerospace nickel cadmium cells were put on life test in February 1979. Each 5 cell pack contained one cell with a standard sensor signal electrode and one cell with a new heart pacer sensor signal electrode. Testing was discontinued in May 1983 and the signal electrode performance data was studied. It was found that the heart pacer electrode generally provided a greater voltage swing over a cycle; that both types of electrodes lost significant sensitivity during life, and that both types of electrodes show great signal variation from cell to cell.

Morrow, G. W.↗

Modulation of Bone Marrow Primary Cell Osteoblastogenesis and Cell Senescence by Mechanical Stimulation

Cell and animal studies conducted onboard the International Space Station and formerly the Shuttle flights have provided groundbreaking data illuminating the deleterious biological response of bone to mechanical unloading. Specifically CDKN1A/p-21 a cell senescence protein, was found to be upregulated in osteoprecursor cells of the femur during 15-day spaceflight, leading to the working hypothesis that CDKN1A/p-21 plays a role in inhibition of bone formation via mechanical regulation. To evaluate this hypothesis, utilizing a p-21 knockout mouse-line and relevant wildtype control, we cultured femoral bone marrow primary cells under unloaded (static) and cyclically stretched loading through a 30 day osteoblastogenesis protocol. Morphologic evaluation of the cultures demonstrated that mechanical stretching aligned the cells and increased the presence of defined focal adhesion expressing talin, integrin αvβ3, and PTK2 protein tyrosine kinase 2, also known as focal adhesion kinase (FAK) in both mouse strains. In corroboration with previous investigations of cell survival signals relation to FAK, our study found that with greater concentration of focal adhesions via stretch stimulation the live cell percentage was significantly higher than the unloaded controls (p-21 knockout line: +49.70%, p*=0.009, wildtype control: +18.14%, p*=.01). Also evaluated was the mineralization and ECM secretion capability of the differentiating cells. Von Kossa staining has shown that in the p-21 knockout cells unloaded cells produce more matrix that the stretch stimulated, however the matrix is unorganized presenting in sporadic nodules covering approximately 30% of the culture area at day 14 (n=6 wells) while the stretch stimulated cultures have less mineralization content the surface area containing mineralized matrix is greater (~68% at day 14). Q-PCR evaluation of the p-21 knockout cells revealed that canonical (β-catenin cascade) and non-canonical wnt11 and downstream planar cell polarity (wnt/PCP) pathway molecule RAC1 are prevalently upregulated with mechanical stimulation. Immunofluorescence for β-catenin and RAC1 showed co-localization at the nuclear membrane of the p-21 knockout cells but not the wildtype (n=1) suggesting that molecular communication via the canonical and wnt/PCP pathway are initiated by mechanical loading and experience regulation along the signaling cascade by CDKN1A/p-21. Future investigations will further elucidate this relationship and provide causal data demonstrating mechanical loading’s modulatory effect on p-21 expression change.

Juran, Cassandra M.↗

Expression of Genes Associated with DNA Damage Sensing in Human Fibroblasts Exposed to Low-dose-rate Gamma Rays

Understanding of the molecular response to low-dose and low-dose-rate radiation exposure is essential for the extrapolation of high-dose radiation risks to those at dose levels relevant to space and other environmental concerns. Most of the reported studies of gene expressions induced by low-dose or low-dose-rate radiation were carried out on exponentially growing cells. In the present study, we analyzed expressions of 84 genes associated with DNA damage sensing using real time PCR in human fibroblasts in mostly G1 phase of the cell cycle. The cells were exposed continuously to gamma rays at a dose rate of 0.8 cGy/hr for 1, 2, 6 or 24 hrs at 37 C throughout the exposure. The total RNA was isolated immediately after the exposure was terminated. Of the 84 genes, only a few showed significant changes of the expression level. Some of the genes (e.g. DDit3 and BTG2) were found to be up or down regulated only after a short period of exposure, while other genes (e.g. PRKDC) displayed a highest expression level at the 24 hr time point. The expression profiles for the exposed cells which had a smaller portion of G1 cells indicated more cell cycle signaling and DNA repair genes either up or down regulated. Interestingly, the panel of genes changed from radiation exposure in G1 cells is different from the panel in cells having less G1 arrest cells. The gene expression profile of the cells responding to low-dose-radiation insult apparently depends on the cell growth stage. The response pathway in G1 cells may differ from that in exponentially growing cells.

Zhang, Ye↗

Distinct abscisic acid signaling pathways for modulation of guard cell versus mesophyll cell potassium channels revealed by expression studies in Xenopus laevis oocytes

Regulation of guard cell ion transport by abscisic acid (ABA) and in particular ABA inhibition of a guard cell inward K(+) current (I(Kin)) is well documented. However, little is known concerning ABA effects on ion transport in other plant cell types. Here we applied patch clamp techniques to mesophyll cell protoplasts of fava bean (Vicia faba cv Long Pod) plants and demonstrated ABA inhibition of an outward K(+) current (I(Kout)). When mesophyll cell protoplast mRNA (mesophyll mRNA) was expressed in Xenopus laevis oocytes, I(Kout) was generated that displayed similar properties to I(Kout) observed from direct analysis of mesophyll cell protoplasts. I(Kout) expressed by mesophyll mRNA-injected oocytes was inhibited by ABA, indicating that the ABA signal transduction pathway observed in mesophyll cells was preserved in the frog oocytes. Co-injection of oocytes with guard cell protoplast mRNA and cRNA for KAT1, an inward K(+) channel expressed in guard cells, resulted in I(Kin) that was similarly inhibited by ABA. However, oocytes co-injected with mesophyll mRNA and KAT1 cRNA produced I(Kin) that was not inhibited by ABA. These results demonstrate that the mesophyll-encoded signaling mechanism could not substitute for the guard cell pathway. These findings indicate that mesophyll cells and guard cells use distinct and different receptor types and/or signal transduction pathways in ABA regulation of K(+) channels.

NASA Discipline Plant Biology↗

Structure, Function, Self-Assembly and Origin of Simple Membrane Proteins

Integral membrane proteins perform such essential cellular functions as transport of ions, nutrients and waste products across cell walls, transduction of environmental signals, regulation of cell fusion, recognition of other cells, energy capture and its conversion into high-energy compounds. In fact, 30-40% of genes in modem organisms codes for membrane proteins. Although contemporary membrane proteins or their functional assemblies can be quite complex, their transmembrane fragments are usually remarkably simple. The most common structural motif for these fragments is a bundle of alpha-helices, but occasionally it could be a beta-barrel. In a series of molecular dynamics computer simulations we investigated self-organizing properties of simple membrane proteins based on these structural motifs. Specifically, we studied folding and insertion into membranes of short, nonpolar or amphiphatic peptides. We also investigated glycophorin A, a peptide that forms sequence-specific dimers, and a transmembrane aggregate of four identical alpha-helices that forms an efficient and selective voltage-gated proton channel was investigated. Many peptides are attracted to water-membrane interfaces. Once at the interface, nonpolar peptides spontaneously fold to a-helices. Whenever the sequence permits, peptides that contain both polar and nonpolar amino also adopt helical structures, in which polar and nonpolar amino acid side chains are immersed in water and membrane, respectively. Specific identity of side chains is less important. Helical peptides at the interface could insert into the membrane and adopt a transmembrane conformation. However, insertion of a single helix is unfavorable because polar groups in the peptide become completely dehydrated upon insertion. The unfavorable free energy of insertion can be regained by spontaneous association of peptides in the membrane. The first step in this process is the formation of dimers, although the most common are aggregates of 4-7 helices. The helices could arrange themselves such that they formed pores capable of transporting ions and small molecules across membranes. Stability of transmembrane aggregates of simple proteins is often only marginal and, therefore, it can be regulated by environmental signals or small sequence modifications in the region of interhelical interactions. A key step in the earliest evolution of membrane proteins was the emergence of selectivity for specific substrates. Many channels could become selective if one or only a few properly chosen amino acids are properly placed along the channel, acting as filters or gates. This is a convenient evolutionary solution because it does not require imposing conditions on the whole sequence.

Pohorille, Andrew↗

Nanoparticle-mediated antagonism of sustained endosomal signaling of the calcitonin receptor-like receptor provides enhanced and persistent relief of oral cancer pain

By improving the delivery and tumor retention of chemotherapeutics, nanomedicines hold potential for cancer treatment. The usefulness of nanoparticle (NP)-encapsulated analgesics for the cancer pain treatment is comparatively unexplored. We investigated whether NPs encapsulating olcegepant (OCP), an antagonist of the calcitonin receptor-like receptor (CLR) for the calcitonin gene-related peptide (CGRP), effectively relieved oral cancer pain in mice. Because persistent endosomal CLR signaling in Schwann cells mediates craniofacial pain, we reasoned that the predisposition of NPs to accumulate in endosomes could be leveraged to effectively relieve oral cancer pain. By expressing biosensors for activated CLR, Gα proteins and β-arrestins in HEK293T and Schwann cells, we found that CGRP activates CLR signaling first at the plasma membrane and then in early, late and recycling endosomes and the cis- and trans-Golgi apparatus. We synthesized biocompatible NPs encapsulating OCP and fluorophores by integrating hydrophobic ion pairing nanoformulation with Flash NanoPrecipitation. NPs slowly released OCP and accumulated in early endosomes, leading to sustained inhibition of endosomal CLR signaling in HEK293T and Schwann cells. Oral cancers were established in mice, which led to heightened pain-like responses. After intra-tumoral injection, NPs were retained in tumors for at least one week. OCP-loaded NPs almost completely reversed allodynia and hyperalgesia for a prolonged period, whereas unencapsulated OCP had small and transient effects. The NP accumulation in endosomal sites of pain signaling, the sustained release of antagonist, and the retention of NPs in tumors explain their beneficial actions. Thus, NP-encapsulation holds promise for the relief of painful cancers that are inadequately treated by opioids.

Calcitonin gene-related peptide↗

Fluorescence activated cell sorting.

An instrument has been developed for sorting biological cells. The cells are rendered differentially fluorescent and incorporated into a small liquid stream illuminated by a laser beam. The cells pass sequentially through the beam, and fluorescent light from the cells gives rise to electrical signals. The stream is broken into a series of uniform size drops downstream of the laser. The cell signals are used to give appropriate electrostatic charges to drops containing the cells. The drops then pass between two charged plates and are deflected to appropriate containers. The system has proved capable of providing fractions containing large numbers of viable cells highly enriched in a particular functional type.

Bonner, W. A.↗