Reduced respiratory neural activity elicits a long-lasting decrease in the CO2 threshold for apnea in anesthetized rats.

Two critical parameters that influence breathing stability are the levels of arterial pCO2 at which breathing ceases and subsequently resumes - termed the apneic and recruitment thresholds (AT and RT, respectively). Reduced respiratory neural activity elicits a chemoreflex-independent, long-lasting increase in phrenic burst amplitude, a form of plasticity known as inactivity-induced phrenic motor facilitation (iPMF). The physiological significance of iPMF is unknown. To determine if iPMF and neural apnea have long-lasting physiological effects on breathing, we tested the hypothesis that patterns of neural apnea that induce iPMF also elicit changes in the AT and RT. Phrenic nerve activity and end-tidal CO2 were recorded in urethane-anesthetized, ventilated rats to quantify phrenic nerve burst amplitude and the AT and RT before and after three patterns of neural apnea that differed in their duration and ability to elicit iPMF: brief intermittent neural apneas, a single brief "massed" neural apnea, or a prolonged neural apnea. Consistent with our hypothesis, we found that patterns of neural apnea that elicited iPMF also resulted in changes in the AT and RT. Specifically, intermittent neural apneas progressively decreased the AT with each subsequent neural apnea, which persisted for at least 60min. Similarly, a prolonged neural apnea elicited a long-lasting decrease in the AT. In both cases, the magnitude of the AT decrease was proportional to iPMF. In contrast, the RT was transiently decreased following prolonged neural apnea, and was not proportional to iPMF. No changes in the AT or RT were observed following a single brief neural apnea. Our results indicate that the AT and RT are differentially altered by neural apnea and suggest that specific patterns of neural apnea that elicit plasticity may stabilize breathing via a decrease in the AT.

Plasticity in respiratory motor neurons in response to reduced synaptic inputs: A form of homeostatic plasticity in respiratory control?

For most individuals, the respiratory control system produces a remarkably stable and coordinated motor output-recognizable as a breath-from birth until death. Very little is understood regarding the processes by which the respiratory control system maintains network stability in the presence of changing physiological demands and network properties that occur throughout life. An emerging principle of neuroscience is that neural activity is sensed and adjusted locally to assure that neurons continue to operate in an optimal range, yet to date, it is unknown whether such homeostatic plasticity is a feature of the neurons controlling breathing. Here, we review the evidence that local mechanisms sense and respond to perturbations in respiratory neural activity, with a focus on plasticity in respiratory motor neurons. We discuss whether these forms of plasticity represent homeostatic plasticity in respiratory control. We present new analyses demonstrating that reductions in synaptic inputs to phrenic motor neurons elicit a compensatory enhancement of phrenic inspiratory motor output, a form of plasticity termed inactivity-induced phrenic motor facilitation (iPMF), that is proportional to the magnitude of activity deprivation. Although the physiological role of iPMF is not understood, we hypothesize that it has an important role in protecting the drive to breathe during conditions of prolonged or intermittent reductions in respiratory neural activity, such as following spinal cord injury or during central sleep apnea.

SOCS3-mediated regulation of inflammatory cytokines in PTEN and p53 inactivated triple negative breast cancer model.

Somatic mutations or deletions of TP53 and PTEN in ductal carcinoma in situ lesions have been implicated in progression to invasive ductal carcinomas. A recent molecular and mutational analysis of breast cancers revealed that inactivation of tumor suppressors, p53 and PTEN, are strongly associated with triple negative breast cancer. In addition, these tumor suppressors have important roles in regulating self-renewal in normal and malignant stem cells. To investigate their role in breast carcinogenesis, we knocked down these genes in human mammary cells and in non-transformed MCF10A cells. p53 and PTEN knockdown synergized to activate pro-inflammatory interleukin-6 (IL6)/Stat3/nuclear factor κB signaling. This resulted in generation of highly metastatic epithelial-to-mesenchymal transition-like cancer stem cells resulting in tumors whose gene expression profile mimicked that found in basal/claudin-low molecular subtype within the triple negative breast tumors. Constitutive activation of this loop in transformed cells was dependent on proteolytic degradation of suppressor of cytokine signaling 3 (SOCS3) resulting in low levels of this protein in basal/claudin-low cell lines and primary tumors. In non-transformed cells, transient activation of the IL6 inflammatory loop induced SOCS3 expression leading to pathway inactivation. In transformed cells, enforced expression of SOCS3 or interfering with IL6 pathway via IL6R blockade inhibited tumor growth and metastasis in mouse xenograft models. Furthermore, circulating tumor cells were significantly reduced in tumor-bearing animals when treated with anti-IL6R antibodies. These studies uncover important connections between inflammation and carcinogenesis and suggest that blocking pro-inflammatory cytokines may be utilized as an attractive strategy to target triple negative breast tumors, which currently lacks molecularly targeted therapies.

Ventilatory behaviors of the toad Bufo marinus revealed by coherence analysis.

Breathing in amphibians is a remarkably complex behavior consisting of irregular breaths that may be taken singly or in bouts that are used to deflate and inflate the lungs. The valves at the two outlets of the buccal cavity (nares and glottis) need to be finely controlled throughout the bout for the expression of these complex respiratory behaviors. In this study, we use a technique based on the calculation of the coherence spectra between respiratory variables (buccal pressure; narial airflow; and lung pressure). Coherence was also used to quantify the effects of chemoreceptor and pulmonary mechanoreceptor input on narial and glottal valve behavior on normoxic, hypoxic, and hypercapnic toads with both intact and bilaterally sectioned pulmonary vagi. We found a significant reduction in narial coherence in hypoxic vagotomized toads indicating that pulmonary mechanoreceptor feedback modulates narial opening duration. An unexpectedly high coherence between Pl and Pb during non-respiratory buccal oscillations in hypercapnic toads indicated more forceful use of the buccal pump. We concluded that the coherence function reveals behaviors that are not apparent through visual inspection of ventilatory time series.

Ventilatory behaviors of the toad Bufo marinus revealed by coherence analysis

Breathing in amphibians is a remarkably complex behavior consisting of irregular breaths that may be taken singly or in bouts that are used to deflate and inflate the lungs. The valves at the two outlets of the buccal cavity (nares and glottis) need to be finely controlled throughout the bout for the expression of these complex respiratory behaviors. In this study, we use a technique based on the calculation of the coherence spectra between respiratory variables (buccal pressure; narial airflow; and lung pressure). Coherence was also used to quantify the effects of chemoreceptor and pulmonary mechanoreceptor input on narial and glottal valve behavior on normoxic, hypoxic, and hypercapnic toads with both intact and bilaterally sectioned pulmonary vagi. We found a significant reduction in narial coherence in hypoxic vagotomized toads indicating that pulmonary mechanoreceptor feedback modulates narial opening duration. An unexpectedly high coherence between Pl and Pb during non-respiratory buccal oscillations in hypercapnic toads indicated more forceful use of the buccal pump. We concluded that the coherence function reveals behaviors that are not apparent through visual inspection of ventilatory time series

Respiratory plasticity: differential actions of continuous and episodic hypoxia and hypercapnia.

The objectives of this paper are: (1) to review advances in our understanding of the mechanisms of respiratory plasticity elicited by episodic versus continuous hypoxia in short to intermediate time domains (min to h); and (2) to present new data suggesting that different patterns of hypercapnia also elicit distinct forms of respiratory plasticity. Episodic, but not continuous hypoxia elicits long-term facilitation (LTF) of respiratory motor output. Phrenic LTF is a serotonin-dependent central neural mechanism that requires: (a) activation of spinal serotonin receptors; and (b) spinal protein synthesis. Continuous and episodic hypercapnia also elicit different mechanisms of plasticity. Continuous, severe hypercapnia (25 min of approximately 10% inspired CO(2)) elicits long-term depression (LTD) of phrenic motor output (-33+/-8% at 60 min post-hypercapnia) in anesthetized rats. In contrast, 3,5 min hypercapnic episodes do not elicit LTD (9+/-17% at 60 min). We hypothesize that the response of respiratory motoneurons to serotonergic and noradrenergic modulation may contribute to pattern sensitivity to hypoxia and hypercapnia.

Invited review: Intermittent hypoxia and respiratory plasticity.

Intermittent hypoxia elicits long-term facilitation (LTF), a persistent augmentation (hours) of respiratory motor output. Considerable recent progress has been made toward an understanding of the mechanisms and manifestations of this potentially important model of respiratory plasticity. LTF is elicited by intermittent but not sustained hypoxia, indicating profound pattern sensitivity in its underlying mechanism. During intermittent hypoxia, episodic spinal serotonin receptor activation initiates cell signaling events, increasing spinal protein synthesis. One associated protein is brain-derived neurotrophic factor, a neurotrophin implicated in several forms of synaptic plasticity. Our working hypothesis is that increased brain-derived neurotrophic factor enhances glutamatergic synaptic currents in phrenic motoneurons, increasing their responsiveness to bulbospinal inspiratory inputs. LTF is heterogeneous among respiratory outputs, differs among experimental preparations, and is influenced by age, gender, and genetics. Furthermore, LTF is enhanced following chronic intermittent hypoxia, indicating a degree of metaplasticity. Although the physiological relevance of LTF remains unclear, it may reflect a general mechanism whereby intermittent serotonin receptor activation elicits respiratory plasticity, adapting system performance to the ever-changing requirements of life.

Phrenic long-term facilitation requires 5-HT receptor activation during but not following episodic hypoxia.

Episodic hypoxia evokes a sustained augmentation of respiratory motor output known as long-term facilitation (LTF). Phrenic LTF is prevented by pretreatment with the 5-hydroxytryptamine (5-HT) receptor antagonist ketanserin. We tested the hypothesis that 5-HT receptor activation is necessary for the induction but not maintenance of phrenic LTF. Peak integrated phrenic nerve activity (integralPhr) was monitored for 1 h after three 5-min episodes of isocapnic hypoxia (arterial PO(2) = 40 +/- 2 Torr; 5-min hyperoxic intervals) in four groups of anesthetized, vagotomized, paralyzed, and ventilated Sprague-Dawley rats [1) control (n = 11), 2) ketanserin pretreatment (2 mg/kg iv; n = 7), and ketanserin treatment 0 and 45 min after episodic hypoxia (n = 7 each)]. Ketanserin transiently decreased integralPhr, but it returned to baseline levels within 10 min. One hour after episodic hypoxia, integralPhr was significantly elevated from baseline in control and in the 0- and 45-min posthypoxia ketanserin groups. Conversely, ketanserin pretreatment abolished phrenic LTF. We conclude that 5-HT receptor activation is necessary to initiate (during hypoxia) but not maintain (following hypoxia) phrenic LTF.

Expression of hypoglossal long-term facilitation differs between substrains of Sprague-Dawley rat.

Long-term facilitation (LTF) is a prolonged, serotonin-dependent augmentation of respiratory motor output following episodic hypoxia. Previous observations lead us to hypothesize that LTF is subject to genetic influences and, as a result, differs between Sprague-Dawley (SD) rats from two vendors, Harlan (H) and Charles River Laboratories/Sasco (CRL/S). Using a blinded experimental design, we recorded integrated phrenic (integralPhr) and hypoglossal neurograms in anesthetized, vagotomized, paralyzed, and ventilated rats. At 60 min following three 5-min hypoxic episodes (Pa(O(2)) = 40 +/- 1 Torr; 5-min hyperoxic intervals), integralPhr was elevated from baseline in both SD substrains (i.e., LTF; P < 0.05). Conversely, hypoglossal LTF was present in CRL/S but not H rats (P < 0.05 between substrains). Serotonin immunoreactivity within the hypoglossal nucleus was not different between H and CRL/S rats. We conclude that the expression of hypoglossal LTF differs between SD rat substrains, indicating a difference in their genetic predisposition to neural plasticity.

Episodic but not continuous hypoxia elicits long-term facilitation of phrenic motor output in rats.

1. Intermittent hypoxia elicits long-term facilitation (LTF) of phrenic motor output in anaesthetized rats. We tested the hypothesis that an equal cumulative duration of continuous hypoxia would not elicit phrenic LTF. 2. Integrated phrenic nerve activity was recorded in urethane-anaesthetized, vagotomized, paralysed and ventilated rats exposed to: (1) 3 X 3 min hypoxic episodes (inspired O2 fraction (FI, O2) = 0.11) separated by 5 min hyperoxia (FI,O2 = 0.5; n = 6), (2) 9 min continuous hypoxia (n = 6), or (3) 20 min continuous hypoxia (n = 7). Isocapnia was maintained throughout the protocol. 3. Consistent with previous studies, phrenic amplitude was significantly elevated for at least 1 h following intermittent hypoxia (78 +/- 15% 60 min post-hypoxia; P < 0.05) with an associated increase in burst frequency (11 +/- 2.1 bursts min-1; P < 0.05). In contrast, 9 or 20 min continuous hypoxia did not elicit LTF of either phrenic amplitude (4.7 +/- 5.1 and 10.1 +/- 10.2% 60 min post-hypoxia, respectively; P > 0.05) or frequency (4.6 +/- 1.3 and 5.1 +/- 2 bursts min-1 60 min post-hypoxia, respectively; P > 0.05). 4. The results indicate that hypoxia-induced long-term facilitation of phrenic motor output is sensitive to the pattern of hypoxic exposure in anaesthetized rats.

Long term facilitation of phrenic motor output.

Episodic hypoxia or electrical stimulation of carotid chemoafferent neurons elicits a sustained, serotonin-dependent augmentation of respiratory motor output known as long term facilitation (LTF). The primary objectives of this paper are to provide an updated review of the literature pertaining to LTF, to investigate the influence of selected variables on LTF via meta-analysis of a large data set from LTF experiments on anesthetized rats, and to propose an updated mechanism of LTF. LTF has been demonstrated in anesthetized and awake experimental preparations, and can be evoked in some human subjects during sleep. The mechanism underlying LTF requires episodic chemoafferent stimulation, and is not elicited by similar cumulative durations of sustained hypoxia. Meta-analysis of phrenic nerve responses following episodic hypoxia in 63 experiments on anesthetized rats (conducted by four investigators over a period of several years) indicates that phrenic LTF magnitude correlates with peak phrenic responses during hypoxia and hypercapnia, but not with the level of hypoxia during episodic exposures. Potential mechanisms underlying these relationships are discussed, and currently available data are synthesized into an updated mechanistic model of LTF. In this model, we propose that LTF arises predominantly from episodic activation of serotonergic receptors on phrenic motoneurons, activating intracellular kinases and, thus, phosphorylating and potentiating ionic currents associated with the glutamate receptors that mediate respiratory drive.

Microgravity culture reduces apoptosis and increases the differentiation of a human colorectal carcinoma cell line.

Our hypothesis is that rotation increases apoptosis in standard tissue culture medium at shear stresses of greater than approximately 0.3 dyn/cm2. Human MIP-101 poorly differentiated colorectal carcinoma cells were cultured for 6 d in complete medium in monolayers, on Teflon-coated nonadherent surfaces (static three-dimensional [3D]) or in rotating 3D cultures either in microgravity in low-earth orbit (3D microg) or in unit gravity on the ground (3D 1g). Apoptosis (determined morphologically), proliferation (by MIB1 staining), and the expression of epidermal growth-factor receptor (EGF-R), TGF-alpha, or TGF-beta were assessed by immunohistochemistry, while the expression of the differentiation marker carcinoembryonic antigen (CEA) was assessed on Western blots. Over the course of 6 d, static 3D cultures displayed the highest rates of proliferation and lowest apoptosis. This was associated with high EGF-R, TGF-alpha, and TGF-beta expression which was greater than that of a monolayer culture. Both rotated 3D lg and 3D microg cultures displayed lower expression of EGF-R, TGF-alpha, or TGF-beta and proliferation than that of monolayer or static 3D cultures. However, rotated 3D microg displayed significantly less apoptosis and greater CEA expression than rotated 3D 1g cultures. When rotated cultures of MIP-101 cells were grown uncler static conditions for another 3 d, proliferation increased and apoptosis decreased. Thus, rotation appears to increase apoptosis and decrease proliferation, whereas static 3D cultures in either unit or microgravity have less apoptosis, and reduced rotation in microgravity increases CEA expression.

S-Nitrosocysteine increases palmitate turnover on Ha-Ras in NIH 3T3 cells.

Ha-Ras is modified by isoprenoid on Cys(186) and by reversibly attached palmitates at Cys(181) and Cys(184). Ha-Ras loses 90% of its transforming activity if Cys(181) and Cys(184) are changed to serines, implying that palmitates make important contributions to oncogenicity. However, study of dynamic acylation is hampered by an absence of methods for acutely manipulating Ha-Ras palmitoylation in living cells. S-nitrosocysteine (SNC) and, to a more modest extent, S-nitrosoglutathione were found to rapidly increase [(3)H]palmitate incorporation into cellular or oncogenic Ha-Ras in NIH 3T3 cells. In contrast, SNC decreased [(3)H]palmitate labeling of the transferrin receptor and caveolin. SNC accelerated loss of [(3)H]palmitate from Ha-Ras, implying that SNC stimulated deacylation and permitted subsequent reacylation of Ha-Ras. SNC also decreased Ha-Ras GTP binding and inhibited phosphorylation of the kinases ERK1 and ERK2 in NIH 3T3 cells. Thus, SNC altered two important properties of Ha-Ras activation state and lipidation. These results identify SNC as a new tool for manipulating palmitate turnover on Ha-Ras and for studying requirements of repalmitoylation and the relationship between palmitate cycling, membrane localization, and signaling by Ha-Ras.

A non-farnesylated Ha-Ras protein can be palmitoylated and trigger potent differentiation and transformation.

Ha-Ras undergoes post-translational modifications (including attachment of farnesyl and palmitate) that culminate in localization of the protein to the plasma membrane. Because palmitate is not attached without prior farnesyl addition, the distinct contributions of the two lipid modifications to membrane attachment or biological activity have been difficult to examine. To test if palmitate is able to support these crucial functions on its own, novel C-terminal mutants of Ha-Ras were constructed, retaining the natural sites for palmitoylation, but replacing the C-terminal residue of the CAAX signal for prenylation with six lysines. Both the Ext61L and ExtWT proteins were modified in a dynamic fashion by palmitate, without being farnesylated; bound to membranes modestly (40% as well as native Ha-Ras); and retained appropriate GTP binding properties. Ext61L caused potent transformation of NIH 3T3 cells and, unexpectedly, an exaggerated differentiation of PC12 cells. Ext61L with the six lysines but lacking palmitates was inactive. Thus, farnesyl is not needed as a signal for palmitate attachment or removal, and a combination of transient palmitate modification and basic residues can support Ha-Ras membrane binding and two quite different biological functions. The roles of palmitate can therefore be independent of and distinct from those of farnesyl. Reciprocally, if membrane association can be sustained largely through palmitates, farnesyl is freed to interact with other proteins.

Three-dimensional culture of bovine chondrocytes in rotating-wall vessels.

The Rotating-Wall Vessel (RWV) was used to culture chondrocytes for 36 d to observe the influence of low-shear and quiescent culture conditions allowing three-dimensional freedom on growth, differentiation, and extracellular matrix formation. Chondrocytes were freshly isolated from bovine cartilage and placed into the RWV with Cytodex-3 microcarriers. Nonadherent petri dishes were initiated with microcarriers as representative of standard culture conditions. In the RWV, large three-dimensional aggregates (5-7 mm) were formed in suspension. In addition, a large sheet of matrix adhered to the oxygenator core and vessel endcaps. Petri dish culture resulted in the formation of sheets of chondrocytes with no matrix production. Immunocytochemical analyses on histologic sections of tissue obtained from the RWV and the petri dish controls were performed with antibodies against fibronectin, collagen II, chondroitin-4-sulfate, chondroitin-6-sulfate, and vimentin. Results demonstrated increased signal in the RWV material while the petri dishes demonstrated a slight decrease in signal. In addition, differentiated chondrocytes were observed in sections of RWV material through 36 d, while few were observed in the sections of petri dish material. These results indicate that the unique conditions provided by the RWV afford access to cellular processes that signify the initiation of differentiation as well as production of normal matrix material.

Establishment of a three-dimensional human prostate organoid coculture under microgravity-simulated conditions: evaluation of androgen-induced growth and PSA expression.

A novel in vitro human prostate cancer model was established by using a coculture technique in which isolated human prostate fibroblasts were observed to grow as a mixed culture with isolated human prostate cancer cells (LNCaP) on microcarrier beads under microgravity-simulated conditions. This model appears to be promising and deserves further exploration because: (a) cocultured human prostate fibroblasts and cancer epithelial cells appear to undergo patterns of histogenesis similar to those observed in human prostate tumors and (b) unlike the conventional cell culture on plastic dishes, cocultured human prostate fibroblasts and LNCaP cells in microgravity-simulated conditions responded to the inductive signals of growth and differentiation from dihydrotestosterone in a manner similar to that observed in the in vivo condition. These results offer an opportunity to examine molecular mechanisms of cellular signaling in response to androgen stimulation during normal and aberrant human prostate development. The microgravity-simulated three-dimensional prostate epithelial cell culture with prostate fibroblasts can be further explored as an ideal in vitro model for the study of normal and neoplastic prostate development. This model could also be adopted as a drug screening program for the discovery of novel therapeutic agents in the treatment of human prostate cancer and benign hyperplastic growth.

Changes in gravity inhibit lymphocyte locomotion through type I collagen.

Immunity relies on the circulation of lymphocytes through many different tissues including blood vessels, lymphatic channels, and lymphoid organs. The ability of lymphocytes to traverse the interstitium in both nonlymphoid and lymphoid tissues can be determined in vitro by assaying their capacity to locomote through Type I collagen. In an attempt to characterize potential causes of microgravity-induced immunosuppression, we investigated the effects of simulated microgravity on human lymphocyte function in vitro using a specialized rotating-wall vessel culture system developed at the Johnson Space Center. This very low shear culture system randomizes gravitational vectors and provides an in vitro approximation of microgravity. In the randomized gravity of the rotating-wall vessel culture system, peripheral blood lymphocytes did not locomote through Type I collagen, whereas static cultures supported normal movement. Although cells remained viable during the entire culture period, peripheral blood lymphocytes transferred to unit gravity (static culture) after 6 h in the rotating-wall vessel culture system were slow to recover and locomote into collagen matrix. After 72 h in the rotating-wall vessel culture system and an additional 72 h in static culture, peripheral blood lymphocytes did not recover their ability to locomote. Loss of locomotory activity in rotating-wall vessel cultures appears to be related to changes in the activation state of the lymphocytes and the expression of adhesion molecules. Culture in the rotating-wall vessel system blunted the ability of peripheral blood lymphocytes to respond to polyclonal activation with phytohemagglutinin. Locomotory response remained intact when peripheral blood lymphocytes were activated by anti-CD3 antibody and interleukin-2 prior to introduction into the rotating-wall vessel culture system. Thus, in addition to the systemic stress factors that may affect immunity, isolated lymphocytes respond to gravitational changes by ceasing locomotion through model interstitium. These in vitro investigations suggest that microgravity induces non-stress-related changes in cell function that may be critical to immunity. Preliminary analysis of locomotion in true microgravity revealed a substantial inhibition of cellular movement in Type I collagen. Thus, the rotating-wall vessel culture system provides a model for analyzing the microgravity-induced inhibition of lymphocyte locomotion and the investigation of the mechanisms related to lymphocyte movement.

Patient satisfaction and service quality in the formation of customers' future purchase intentions in competitive health service settings.

The following study provides evidence that the relationship between quality perceptions and satisfaction judgements in the formation of future purchase intentions may be very different in health service settings relative to other service settings. The study investigates Taylor and Baker's (1994) assertion that satisfaction judgements moderate the quality-->purchase intention relationship by testing the research model in both for-profit and not-for-profit hospital settings. The results of this study first support the growing view that satisfaction judgements are more closely related to outcome behaviors than quality perceptions in hospital settings. The results further support the assertion that the formation of important consumer outcomes, such as future purchase intentions, appears to be different for health services. Thus, health service managers are cautioned to empirically test models in the literature specific to their own competitive setting. The managerial and research implications of the reported study are presented and discussed.