Intrinsic pH-Sensitivity of Cells in the Retrotrapezoid Nucleus: Possible Role of Glia in Respiratory Drive

An increase in carbon dioxide (CO2) and protons (H+) are the primary signals for breathing. Cells that sense changes in CO2/H+ levels and increase breathing accordingly are located in a region of the caudal medulla oblongata called the retrotrapezoid nucleus (RTN). Specifically, select RTN neurons are intrinsically pH sensitive and send excitatory projections to the respiratory rhythm generator to drive breathing. Glial cells in the RTN are thought to contribute to this respiratory drive, possibly by releasing ATP in response to increases in CO2/H+ levels. However, pH sensitivity of RTN glial cells has yet to be determined. Therefore, the goal of my thesis is to determine if acutely dissociated RTN cells can respond to changes in pH in isolation. To make this determination I used ratiometric fluorescent microscopy to measure intracellular calcium in dissociated RTN cells during changes in bath pH. I found that a small percentage of RTN cells (16%) respond to bath acidification from pH 7.3 to pH 6.9 with an increase in fluorescence indicating an increase in intracellular calcium. Preliminary electrophysiological findings suggest that responsive cells are unable to make action potentials, thus suggesting their identity to be glia. These results indicate that a subset of pH sensitive cells in the RTN are intrinsically pH sensitive and that glia cells may possibly play a role in central chemoreception.

Intrinsic oxidative stress in cancer cells: A biochemical basis for therapeutic selectivity using ROS -generating agents

A major goal of chemotherapy is to selectively kill cancer cells while minimizing toxicity to normal cells. Identifying biological differences between cancer and normal cells is essential in designing new strategies to improve therapeutic selectivity. Superoxide dismutases (SOD) are crucial antioxidant enzymes required for the elimination of superoxide (O2·− ), a free radical produced during normal cellular metabolism. Previous studies in our laboratory demonstrated that 2-methoxyestradiol (2-ME), an estradiol derivative, inhibits the function of SOD and selectively kills human leukemia cells without exhibiting significant cytotoxicity in normal lymphocytes. The present work was initiated to examine the biochemical basis for the selective anticancer activity of 2-ME. Investigations using two-parameter flow cytometric analyses and ROS scavengers established that O2·− is a primary and essential mediator of 2-ME-induced apoptosis in cancer cells. In addition, experiments using SOD overexpression vectors and SOD knockout cells found that SOD is a critical target of 2-ME. Importantly, the administration of 2-ME resulted in the selective accumulation of O 2·− and apoptosis in leukemia and ovarian cancer cells. The preferential activity of 2-ME was found to be due to increased intrinsic oxidative stress in these cancer cells versus their normal counterparts. This intrinsic oxidative stress was associated with the upregulation of the antioxidant enzymes SOD and catalase as a mechanism to cope with the increase in ROS. Furthermore, oxygen consumption experiments revealed that normal lymphocytes decrease their respiration rate in response to 2-ME-induced oxidative stress, while human leukemia cells seem to lack this regulatory mechanism. This leads to an uncontrolled production of O2·−, severe accumulation of ROS, and ultimately ROS-mediated apoptosis in leukemia cells treated with 2-ME. The biochemical differences between cancer and normal cells identified here provide a basis for the development of drug combination strategies using 2-ME with other ROS-generating agents to enhance anticancer activity. The effectiveness of such a combination strategy in killing cancer cells was demonstrated by the use of 2-ME with agents/modalities such as ionizing radiation and doxorubicin. Collectively, the data presented here strongly suggests that 2-ME may have important clinical implications for the selective killing of cancer cells. ^

Regulation of intracellular pH in cnidarians: response to acidosis in Anemonia viridis

The regulation of intracellular pH (pHi) is a fundamental aspect of cell physiology that has received little attention in studies of the phylum Cnidaria, which includes ecologically important sea anemones and reef-building corals. Like all organisms, cnidarians must maintain pH homeostasis to counterbalance reductions in pHi, which can arise because of changes in either intrinsic or extrinsic parameters. Corals and sea anemones face natural daily changes in internal fluids, where the extracellular pH can range from 8.9 during the day to 7.4 at night. Furthermore, cnidarians are likely to experience future CO2-driven declines in seawater pH, a process known as ocean acidification. Here, we carried out the first mechanistic investigation to determine how cnidarian pHi regulation responds to decreases in extracellular and intracellular pH. Using the anemone Anemonia viridis, we employed confocal live cell imaging and a pH-sensitive dye to track the dynamics of pHi after intracellular acidosis induced by acute exposure to decreases in seawater pH and NH4Cl prepulses. The investigation was conducted on cells that contained intracellular symbiotic algae (Symbiodinium sp.) and on symbiont-free endoderm cells. Experiments using inhibitors and Na-free seawater indicate a potential role of Na/H plasma membrane exchangers (NHEs) in mediating pHi recovery following intracellular acidosis in both cell types. We also measured the buffering capacity of cells, and obtained values between 20.8 and 43.8 mM per pH unit, which are comparable to those in other invertebrates. Our findings provide the first steps towards a better understanding of acid-base regulation in these basal metazoans, for which information on cell physiology is extremely limited.

Pushover analysis for the seismic response prediction of cable-stayed bridges under multi-directional excitation

Cable-stayed bridges represent nowadays key points in transport networks and their seismic behavior needs to be fully understood, even beyond the elastic range of materials. Both nonlinear dynamic (NL-RHA) and static (pushover) procedures are currently available to face this challenge, each with intrinsic advantages and disadvantages, and their applicability in the study of the nonlinear seismic behavior of cable-stayed bridges is discussed here. The seismic response of a large number of finite element models with different span lengths, tower shapes and class of foundation soil is obtained with different procedures and compared. Several features of the original Modal Pushover Analysis (MPA) are modified in light of cable-stayed bridge characteristics, furthermore, an extension of MPA and a new coupled pushover analysis (CNSP) are suggested to estimate the complex inelastic response of such outstanding structures subjected to multi-axial strong ground motions.

Integration of multiple instructive cues by neural crest stem cells reveals cell-intrinsic biases in relative growth factor responsiveness

Growth factors can influence lineage determination of neural crest stem cells (NCSCs) in an instructive manner, in vitro. Because NCSCs are likely exposed to multiple signals in vivo, these findings raise the question of how stem cells would integrate such combined influences. Bone morphogenetic protein 2 (BMP2) promotes neuronal differentiation and glial growth factor 2 (GGF2) promotes glial differentiation; if NCSCs are exposed to saturating concentrations of both factors, BMP2 appears dominant. By contrast, if the cells are exposed to saturating concentrations of both BMP2 and transforming growth factor β1 (which promotes smooth muscle differentiation), the two factors appear codominant. Sequential addition experiments indicate that NCSCs require 48–96 hrs in GGF2 before they commit to a glial fate, whereas the cells commit to a smooth muscle fate within 24 hr in transforming growth factor β1. The delayed response to GGF2 does not reflect a lack of functional receptors; however, because the growth factor induces rapid mitogen-activated protein kinase phosphorylation in naive cells. Furthermore, GGF2 can attenuate induction of the neurogenic transcription factor mammalian achaete-scute homolog 1, by low doses of BMP2. This short-term antineurogenic influence of GGF2 is not sufficient for glial lineage commitment, however. These data imply that NCSCs exhibit cell-intrinsic biases in the timing and relative dosage sensitivity of their responses to instructive factors that influence the outcome of lineage decisions in the presence of multiple factors. The relative delay in glial lineage commitment, moreover, apparently reflects successive short-term and longer-term actions of GGF2. Such a delay may help to explain why glia normally differentiate after neurons, in vivo.

δ-Tonoplast intrinsic protein defines unique plant vacuole functions

Plant cell vacuoles may have either storage or degradative functions. Vegetative storage proteins (VSPs) are synthesized in response to wounding and to developmental switches that affect carbon and nitrogen sinks. Here we show that VSPs are stored in a unique type of vacuole that is derived from degradative central vacuoles coincident with insertion of a new tonoplast intrinsic protein (TIP), δ-TIP, into their membranes. This finding demonstrates a tight coupling between the presence of δ-TIP and acquisition of a specialized storage function and indicates that TIP isoforms may determine vacuole identity.

Intrinsic responses to Borna disease virus infection of the central nervous system

Immune cells invading the central nervous system (CNS) in response to Borna disease virus (BDV) antigens are central to the pathogenesis of Borna disease (BD). We speculate that the response of the resident cells of the brain to infection may be involved in the sensitization and recruitment of these inflammatory cells. To separate the responses of resident cells from those of cells infiltrating from the periphery, we used dexamethasone to inhibit inflammatory reactions in BD. Treatment with dexamethasone prevented the development of clinical signs of BD, and the brains of treated animals showed no neuropathological lesions and a virtual absence of markers of inflammation, cell infiltration, or activation normally seen in the CNS of BDV-infected rats. In contrast, treatment with dexamethasone exacerbated the expression of BDV RNA, which was paralleled by a similarly elevated expression of mRNAs for egr-1, c-fos, and c-jun. Furthermore, dexamethasone failed to inhibit the increase in expression of mRNAs for tumor necrosis factor α, macrophage inflammatory protein 1β, interleukin 6, and mob-1, which occurs in the CNS of animals infected with BDV. Our findings suggest that these genes, encoding transcription factors, chemokines, and proinflammatory cytokines, might be directly activated in CNS resident cells by BDV. This result supports the hypothesis that the initial phase of the inflammatory response to BDV infection in the brain may be dependent upon virus-induced activation of CNS resident cells.

The intrinsic radioresistance of glioblastoma-derived cell lines is associated with a failure of p53 to induce p21BAX expression

Radiation is the primary modality of therapy for all commonly occurring malignant brain tumors, including medulloblastoma and glioblastoma. These two brain tumors, however, have a distinctly different response to radiation therapy. Medulloblastoma is very sensitive to radiation therapy, whereas glioblastoma is highly resistant, and the long-term survival of medulloblastoma patients exceeds 50%, while there are few long-term survivors among glioblastoma patients. p53-mediated apoptosis is thought to be an important mechanism mediating the cytotoxic response of tumors to radiotherapy. In this study, we compared the response to radiation of five cell lines that have wild-type p53: three derived from glioblastoma and two derived from medulloblastoma. We found that the medulloblastoma-derived cell lines underwent extensive radiation-induced apoptotic cell death, while those from glioblastomas did not exhibit significant radiation-induced apoptosis. p53-mediated induction of p21BAX is thought to be a key component of the pathway mediating apoptosis after the exposure of cells to cytotoxins, and the expression of mRNA encoding p21BAX was correlated with these cell lines undergoing radiation-induced apoptosis. The failure of p53 to induce p21BAX expression in glioblastoma-derived cell lines is likely to be of biologic significance, since inhibition of p21BAX induction in medulloblastoma resulted in a loss of radiation-induced apoptosis, while forced expression of p21BAX in glioblastoma was sufficient to induce apoptosis. The failure of p53 to induce p21BAX in glioblastoma-derived cell lines suggests a distinct mechanism of radioresistance and may represent a critical factor in determining therapeutic responsiveness to radiation in glioblastomas.

Modular organization of intrinsic connections associated with spectral tuning in cat auditory cortex

Many response properties in primary auditory cortex (AI) are segregated spatially and organized topographically as those in primary visual cortex. Intensive study has not revealed an intrinsic, anatomical organizing principle related to an AI functional topography. We used retrograde anatomic tracing and topographic physiologic mapping of acoustic response properties to reveal long-range (≥1.5 mm) convergent intrinsic horizontal connections between AI subregions with similar bandwidth and characteristic frequency selectivity. This suggests a modular organization for processing spectral bandwidth in AI.

Subthreshold facilitation and suppression in primary visual cortex revealed by intrinsic signal imaging.

Neurons in primary visual cortex (area 17) respond vigorously to oriented stimuli within their receptive fields; however, stimuli presented outside the suprathreshold receptive field can also influence their responses. Here we describe a fundamental feature of the spatial interaction between suprathreshold center and subthreshold surround. By optical imaging of intrinsic signals in area 17 in response to a stimulus border, we show that a given stimulus generates activity primarily in iso-orientation domains, which extend for several millimeters across the cortical surface in a manner consistent with the architecture of long-range horizontal connections in area 17. By mapping the receptive fields of single neurons and imaging responses from the same cortex to stimuli that include or exclude the aggregate suprathreshold receptive field, we show that intrinsic signals strongly reveal the subthreshold surround contribution. Optical imaging and single-unit recording both demonstrate that the relative contrast of center and surround stimuli regulates whether surround interactions are facilitative or suppressive: the same surround stimulus facilitates responses when center contrast is low, but suppresses responses when center contrast is high. Such spatial interactions in area 17 are ideally suited to contribute to phenomena commonly regarded as part of "higher-level" visual processing, such as perceptual "popout" and "filling-in."

The CD19 signal transduction molecule is a response regulator of B-lymphocyte differentiation.

The phenotypes of CD19-deficient (CD19-/-) mice, and human CD19-transgenic (hCD19TG) mice that overexpress CD19 indicate that CD19 is a response regulator of B-lymphocyte surface receptor signaling. To further characterize the function of CD19 during B-cell differentiation, humoral immune responses to a T-cell-independent type 1 [trinitrophenyl-lipopolysaccharide (TNP-LPS)], a T-cell-independent type 2 [dinitrophenyl (DNP)-Ficoll], and a T-cell-dependent [DNP-keyhole limpet hemocyanin (KLH)] antigen were assessed in CD19-/- and hCD19TG mice. B cells from CD19-/- mice differentiated and underwent immunoglobulin isotype switching in vitro in response to mitogens and cytokines. In vivo, CD19-/- mice generated humoral responses to TNP-LPS and DNP-KLH that were dramatically lower than those of wild-type littermates. Surprisingly, the humoral response to DNP-Ficoll was significantly greater in CD19-/- mice. In contrast, hCD19TG mice were hyperresponsive to TNP-LPS and DNP-KLH immunization but were hyporesponsive to DNP-Ficoll. These results demonstrate that CD19 is not required for B-cell differentiation and isotype switching but serves as a response regulator which modulates B-cell differentiation. Since humoral responses to both T-cell-dependent and T-cell-independent antigens were similarly affected by alterations in CD19 expression, these differences are most likely to result from intrinsic changes in B-cell function rather than from the selective disruption of B-cell interactions with T cells.

Pubovaginal sling versus transurethral Macroplastique for stress urinary incontinence and intrinsic sphincter deficiency: a prospective randomised controlled trial

Objective To compare the pubovaginal sling and transurethral Macroplastique in the treatment of female stress urinary incontinence (SUI) and intrinsic sphincter deficiency (ISD). Design A prospective randomised controlled trial comparing two surgical treatments for SUI and ISD. Setting Tertiary referral urogynaecology unit in Australia. Population Women with SUI and ISD who were suitable for either surgical technique. Methods Forty-five women with SUI and ISD were randomly allocated the pubovaginal sling (n = 22) or transurethral Macroplastique (n = 23). Subjective and objective success rates, patient satisfaction and cost measurements at six months and one year following surgery were the primary outcome measures. A telephone questionnaire survey was performed at a mean follow up period of 62 months (43-71). Main outcome measure Comparison of success rates, complications and costs. Results The symptomatic and patient satisfaction success rates were similar following the sling and Macroplastique with the objective success rate being significantly greater (P < 0.001) following the sling (81% vs 9%). Macroplastique had significantly lower morbidity but was more expensive than the sling (P < 0.001). Response rate at 62 months follow up was 60% in both groups with the sling group reporting better continence success (69% vs 21%) and satisfaction rates (69% vs 29%, P = 0.057). Conclusions The pubovaginal sling was more effective and economical than transurethral Macroplastique for the treatment of SUI and ISD. However, transurethral Macroplastique remains an appropriate treatment in selected cases of SUI and ISD.

Developmental changes in expression of GABA(A) receptor-channels in rat intrinsic cardiac ganglion neurones

The effects of gamma-aminobutyric acid (GABA) on the electrophysiological properties of intracardiac neurones were investigated in the intracardiac ganglion plexus in situ and in dissociated neurones from neonatal, juvenile and adult rat hearts. Focal application of GABA evoked a depolarizing, excitatory response in both intact and dissociated intracardiac ganglion neurones. Under voltage clamp, both GABA and muscimol elicited inward currents at -60 mV in a concentration-dependent manner. The fast, desensitizing currents were mimicked by the GABA(A) receptor agonists muscimol and taurine, and inhibited by the GABA(A) receptor antagonists, bicuculline and picrotoxin. The GABA(A0) antagonist (1,2,5,6-tetrahydropyridin-4-yl)methyl phosphonic acid (TPMPA), had no effect on GABA-induced currents, suggesting that GABA(A) receptor-channels mediate the response. The GABA-evoked current amplitude recorded from dissociated neurones was age dependent whereby the peak current density measured at -100 mV was similar to 20 times higher for intracardiac neurones obtained from neonatal rats (P2-5) compared with adult rats (P45-49). The decrease in GABA sensitivity occurred during the first two postnatal weeks and coincides with maturation of the sympathetic innervation of the rat heart. Immunohistochemical staining using antibodies against GABA demonstrate the presence of GABA in the intracardiac ganglion plexus of the neonatal rat heart. Taken together, these results suggest that GABA and taurine may act as modulators of neurotransmission and cardiac function in the developing mammalian intrinsic cardiac nervous system.

Hebbian and homeostatic plasticity mechanisms in regular spiking and intrinsic bursting cells of cortical layer 5

Layer 5 contains the major projection neurons of the neocortex and is composed of two major cell types: regular spiking (RS) cells, which have cortico-cortical projections, and intrinsic bursting cells (IB), which have subcortical projections. Little is known about the plasticity processes and specifically the molecular mechanisms by which these two cell classes develop and maintain their unique integrative properties. In this study, we find that RS and IB cells show fundementally different experience-dependent plasticity processes and integrate Hebbian and homeostatic components of plasticity differently. Both RS and IB cells showed TNFα-dependent homeostatic plasticity in response to sensory deprivation, but IB cells were capable of a much faster synaptic depression and homeostatic rebound than RS cells. Only IB cells showed input-specific potentiation that depended on CaMKII autophosphorylation. Our findings demonstrate that plasticity mechanisms are not uniform within the neocortex, even within a cortical layer, but are specialized within subcircuits.

Optical imaging of retina in response to grating stimuli in cats

We examined the intrinsic signals in response to grating stimuli in order to determine whether the light-evoked intrinsic signals of the retina are due to changes in the photoreceptor activities induced by the image projected on to the retina or are due to neural activities of the inner retina. The retinas of the left eye of 12 cats under general anesthesia were examined by a functional imaging fundus camera. Near infrared light was used to monitor the reflectance changes (RCs) of the retina. Vertical grating were used to stimulate the retina at 4 Hz. The spatial frequencies of the gratings were 0.05, 0.11, 0.22, 0.43, 0.86, 1.73, and 3.46 cycles/degree (cpd). Ten images were averaged and used to analyze the RCs to obtain the peak value (PV) of a two dimensional fast Fourier transfer of the RCs. The wavefront aberrations (WA) were measured with a compact wavefront aberrometer and the spatial modulation transfer function (MTF) of the eye was calculated. The retinal reflectance image had a grating pattern. The PV of the spatial sensitivity curve was highest at low spatial frequencies (0.05 and 0.11 cpd), and the sensitivity decreased steeply with an increase in the spatial frequency. RCs were not detectable at 3.46 cpd. The MTF decreased gradually with increases in the spatial frequencies and was 0.68 at 3.46 cpd. The reflectance pattern of the retinal intrinsic signal elicited by grating stimuli of different spatial frequencies was different from that of the MTF. This suggests that the intrinsic signal represents not only the response of the photoreceptors but also other neuronal or vascular changes in the retina.