Adenylate Cyclase Toxin Promotes Internalisation of Integrins and Raft Components and Decreases Macrophage Adhesion Capacity

10 p.

The Voltage-Gated Sodium Channel Na(v)1.8 Is Expressed in Human Sperm

The role of Na+ fluxes through voltage-gated sodium channels in the regulation of sperm cell function remains poorly understood. Previously, we reported that several genes encoding voltage-gated Na+ channels were expressed in human testis and mature spermatozoa. In this study, we analyzed the presence and function of the TTX-resistant VGSC a subunit Na(v)1.8 in human capacitated sperm cells. Using an RT-PCR assay, we found that the mRNA of the gene SCN10A, that encode Na-v1.8, was abundantly and specifically expressed in human testis and ejaculated spermatozoa. The Na-v1.8 protein was detected in capacitated sperm cells using three different specific antibodies against this channel. Positive immunoreactivity was mainly located in the neck and the principal piece of the flagellum. The presence of Na-v1.8 in sperm cells was confirmed by Western blot. Functional studies demonstrated that the increases in progressive motility produced by veratridine, a voltage-gated sodium channel activator, were reduced in sperm cells preincubated with TTX (10 mu M), the Na-v1.8 antagonist A-803467, or a specific Na-v1.8 antibody. Veratridine elicited similar percentage increases in progressive motility in sperm cells maintained in Ca2+-containing or Ca2+-free solution and did not induce hyperactivation or the acrosome reaction. Veratridine caused a rise in sperm intracellular Na+, [Na+](i), and the sustained phase of the response was inhibited in the presence of A-803467. These results verify that the Na+ channel Na-v1.8 is present in human sperm cells and demonstrate that this channel participates in the regulation of sperm function.

Functional analysis of cancer-associated EGFR mutants using a cellular assay with YFP-tagged EGFR intracellular domain

Background: The presence of EGFR kinase domain mutations in a subset of NSCLC patients correlates with the response to treatment with the EGFR tyrosine kinase inhibitors gefitinib and erlotinib. Although most EGFR mutations detected are short deletions in exon 19 or the L858R point mutation in exon 21, more than 75 different EGFR kinase domain residues have been reported to be altered in NSCLC patients. The phenotypical consequences of different EGFR mutations may vary dramatically, but the majority of uncommon EGFR mutations have never been functionally evaluated. Results: We demonstrate that the relative kinase activity and erlotinib sensitivity of different EGFR mutants can be readily evaluated using transfection of an YFP-tagged fragment of the EGFR intracellular domain (YFP-EGFR-ICD), followed by immunofluorescence microscopy analysis. Using this assay, we show that the exon 20 insertions Ins770SVD and Ins774HV confer increased kinase activity, but no erlotinib sensitivity. We also show that, in contrast to the common L858R mutation, the uncommon exon 21 point mutations P848L and A859T appear to behave like functionally silent polymorphisms. Conclusion: The ability to rapidly obtain functional information on EGFR variants of unknown relevance using the YFP-EGFR-ICD assay might prove important in the future for the management of NSCLC patients bearing uncommon EGFR mutations. In addition, our assay may be used to determine the response of resistant EGFR mutants to novel second-generation TKIs.

Response of multiple simultaneously recorded macaque area LIP neurons in a memory saccade task

Cells in the lateral intraparietal cortex (LIP) of rhesus macaques respond vigorously and in spatially-tuned fashion to briefly memorized visual stimuli. Responses to stimulus presentation, memory maintenance, and task completion are seen, in varying combination from neuron to neuron. To help elucidate this functional segmentation a new system for simultaneous recording from multiple neighboring neurons was developed. The two parts of this dissertation discuss the technical achievements and scientific discoveries, respectively.

Technology. Simultanous recordings from multiple neighboring neurons were made with four-wire bundle electrodes, or tetrodes, which were adapted to the awake behaving primate preparation. Signals from these electrodes were partitionable into a background process with a 1/f-like spectrum and foreground spiking activity spanning 300-6000 Hz. Continuous voltage recordings were sorted into spike trains using a state-of-the-art clustering algorithm, producing a mean of 3 cells per site. The algorithm classified 96% of spikes correctly when tetrode recordings were confirmed with simultaneous intracellular signals. Recording locations were verified with a new technique that creates electrolytic lesions visible in magnetic resonance imaging, eliminating the need for histological processing. In anticipation of future multi-tetrode work, the chronic chamber microdrive, a device for long-term tetrode delivery, was developed.

Science. Simultaneously recorded neighboring LIP neurons were found to have similar preferred targets in the memory saccade paradigm, but dissimilar peristimulus time histograms, PSTH). A majority of neighboring cell pairs had a difference in preferred directions of under 45° while the trial time of maximal response showed a broader distribution, suggesting homogeneity of tuning with het erogeneity of function. A continuum of response characteristics was present, rather than a set of specific response types; however, a mapping experiment suggests this may be because a given cell's PSTH changes shape as well as amplitude through the response field. Spike train autocovariance was tuned over target and changed through trial epoch, suggesting different mechanisms during memory versus background periods. Mean frequency-domain spike-to-spike coherence was concentrated below 50 Hz with a significant maximum of 0.08; mean time-domain coherence had a narrow peak in the range ±10 ms with a significant maximum of 0.03. Time-domain coherence was found to be untuned for short lags (10 ms), but significantly tuned at larger lags (50 ms).

Pivoting between Calmodulin Lobes Triggered by Calcium in the Kv7.2/Calmodulin Complex

Kv7.2 (KCNQ2) is the principal molecular component of the slow voltage gated M-channel, which strongly influences neuronal excitability. Calmodulin (CaM) binds to two intracellular C-terminal segments of Kv7.2 channels, helices A and B, and it is required for exit from the endoplasmic reticulum. However, the molecular mechanisms by which CaM controls channel trafficking are currently unknown. Here we used two complementary approaches to explore the molecular events underlying the association between CaM and Kv7.2 and their regulation by Ca2+. First, we performed a fluorometric assay using dansylated calmodulin (D-CaM) to characterize the interaction of its individual lobes to the Kv7.2 CaM binding site (Q2AB). Second, we explored the association of Q2AB with CaM by NMR spectroscopy, using N-15-labeled CaM as a reporter. The combined data highlight the interdependency of the N- and C-lobes of CaM in the interaction with Q2AB, suggesting that when CaM binds Ca2+ the binding interface pivots between the N-lobe whose interactions are dominated by helix B and the C-lobe where the predominant interaction is with helix A. In addition, Ca2+ makes CaM binding to Q2AB more difficult and, reciprocally, the channel weakens the association of CaM with Ca2+.

An analysis of the cooperative mechano-sensitive feedback between intracellular signaling, focal adhesion development, and stress fiber contractility

Cells communicate with their external environment via focal adhesions and generate activation signals that in turn trigger the activity of the intracellular contractile machinery. These signals can be triggered by mechanical loading that gives rise to a cooperative feedback loop among signaling, focal adhesion formation, and cytoskeletal contractility, which in turn equilibrates with the applied mechanical loads. We devise a signaling model that couples stress fiber contractility and mechano-sensitive focal adhesion models to complete this above mentioned feedback loop. The signaling model is based on a biochemical pathway where IP3 molecules are generated when focal adhesions grow. These IP3 molecules diffuse through the cytosol leading to the opening of ion channels that disgorge Ca2+ from the endoplasmic reticulum leading to the activation of the actin/myosin contractile machinery. A simple numerical example is presented where a one-dimensional cell adhered to a rigid substrate is pulled at one end, and the evolution of the stress fiber activation signal, stress fiber concentrations, and focal adhesion distributions are investigated. We demonstrate that while it is sufficient to approximate the activation signal as spatially uniform due to the rapid diffusion of the IP3 through the cytosol, the level of the activation signal is sensitive to the rate of application of the mechanical loads. This suggests that ad hoc signaling models may not be able to capture the mechanical response of cells to a wide range of mechanical loading events. © 2011 American Society of Mechanical Engineers.

Mechanisms of H+ modulation of glycinergic response in rat sacral dorsal commissural neurons

Many ionotropic receptors are modulated by extracellular H+. So far, few studies have directly addressed the role of such modulation at synapses. In the present study, we investigated the effects of changes in extracellular pH on glycinergic miniature inhibitory postsynaptic currents (mIPSCs) as well as glycine-evoked currents (I-Gly) in mechanically dissociated spinal neurons with native synaptic boutons preserved. H+ modulated both the mIPSCs and I-Gly, biphasically, although it activated an amiloride-sensitive inward current by itself. Decreasing extracellular pH reversibly inhibited the amplitude of the mIPSCs and I-Gly, while increasing external pH reversibly potentiated these parameters. Blockade of acid-sensing ion channels (ASICs) with amiloride, the selective antagonist of ASICs, or decreasing intracellular pH did not alter the modulatory effect of H+ on either mIPSCs or I-Gly, H+ shifted the EC50 of the glycine concentration-response curve from 49.3 +/- 5.7 muM at external pH 7.4 to 131.5 +/- 8.1 muM at pH 5.5, without altering the Cl- selectivity of the glycine receptor (GlyR), the Hill coefficient and the maximal I-Gly, suggesting a competitive inhibition of I-Gly by H+. Both Zn2+ and H+ inhibited I-Gly. However, H+ induced no further inhibition of I-Gly in the presence of a saturating concentration of Zn2+. In addition, H+ significantly affected the kinetics of glycinergic mIPSCs and I-Gly. It is proposed that H+ and/or Zn2+ compete with glycine binding and inhibit the amplitude of glycinergic mIPSCs and I-Gly. Moreover, binding of H+ induces a global conformational change in GlyR, which closes the GlyR Cl- channel and results in the acceleration of the seeming desensitization of IGly as well as speeding up the decay time constant of glycinergic mIPSCs. However, the deprotonation rate is faster than the unbinding rate of glycine from the GlyR, leading to reactivation of the undesensitized GlyR after washout of agonist and the appearance of a rebound I-Gly. H+ also modulated the glycine cotransmitter, GABA-activated current (I-GABA). Taken together, the results support a 'conformational coupling' model for H+ modulation of the GlyR and suggest that W may act as a novel modulator for inhibitory neurotransmission in the mammalian spinal cord.

Effect of dietary supplementation of vitamins A, D-3, E, and C on yearling rice field eel, Monopterus albus: Serum indices, gonad development, and metabolism of calcium and phosphorus

The objective of this study was to determine the effect of dietary vitamins A, D-3, E, and C on the gonad development, lipid peroxidation, and immune response of yearling rice field eel, Monopterus albus. A 6-wk feeding trial was designed according to an L-16(4(5)) orthogonal design, in which four vitamins, each at four supplementation levels, were arranged. Sixteen diets were mixed with the different vitamin levels and randomly assigned to 16 groups of fish. Increasing dietary vitamin E supplementation level significantly (P <= 0.05) increased the gonadosomatic index and lowered the serum content of malondialdehyde of rice field eel. Increasing dietary vitamin A and C levels also showed similar effect, but the differences were not statistically significant. Serum immunoglobulin M content increased significantly (P <= 0.01) as dietary vitamin C supplementation levels increased. The concentrations of calcium in bones showed significant (P <= 0.05) trend with vitamin D-3 and A supplementation levels, but the bone phosphorus content was not affected by the dietary vitamin levels.

A SERINE KINASE REGULATES INTRACELLULAR-LOCALIZATION OF SPLICING FACTORS IN THE CELL CYCLE

Small nuclear ribonucleoprotein particles (snRNPs) and non-snRNP splicing factors containing a serine/arginine-rich domain (SR proteins) concentrate in 'speckles' in the nucleus of interphase cells(1). It is believed that nuclear speckles act as storage sites for splicing factors while splicing occurs on nascent transcripts(2). Splicing factors redistribute in response to transcription inhibition(3,4) or viral infection(5), and nuclear speckles break down and reform as cells progress through mitosis(6). We have now identified and cloned a kinase, SRPK1, which is regulated by the cell cycle and is specific for SR proteins; this kinase is related to a Caenorhabditis elegans kinase and to the fission yeast kinase Dsk1 (ref. 7). SRPK1 specifically induces the disassembly of nuclear speckles, and a high level of SRPK1 inhibits splicing in vitro. Our results indicate that SRPK1 mag have a central role in the regulatory network for splicing, controlling the intranuclear distribution of splicing factors in interphase cells, and the reorganization of nuclear speckles during mitosis.

Differential regulation of Sciaenops ocellatus viperin expression by intracellular and extracellular bacterial pathogens

Viperin is an antiviral protein that has been found to exist in diverse vertebrate organisms and is involved in innate immunity against the infection of a wide range of viruses. However, it is largely unclear as to the potential role played by viperin in bacterial infection. In this study, we identified the red drum Sciaenops ocellatus viperin gene (SoVip) and analyzed its expression in relation to bacterial challenge. The complete gene of SoVip is 2570 bp in length and contains six exons and five introns. The open reading frame of SoVip is 1065 bp, which is flanked by a 5'-untranslated region (UTR) of 34 bp and a 3'-UTR of 350 bp. The deduced amino acid sequence of SoVip shares extensive identities with the viperins of several fish species and possesses the conserved domain of the radical S-adenosylmethionine superfamily proteins. Expressional analysis showed that constitutive expression of SoVip was relatively high in blood, muscle, brain, spleen, and liver, and low in kidney, gill, and heart. Experimental challenges with poly(I:C) and bacterial pathogens indicated that SoVip expression in liver was significantly upregulated by poly(I:C) and the fish pathogen Edwardsiella tarda but down-regulated by the fish pathogens Listonella anguillarum and Streptococcus iniae. Similar differential induction patterns were also observed at cellular level with primary hepatocytes challenged with E. tarda, L anguillarum, and S. iniae. Infection study showed that all three bacterial pathogens could attach to cultured primary hepatocytes but only E. tarda was able to invade into and survive in hepatocytes. Together these results indicate that SoVip is involved in host immune response during bacterial infection and is differentially regulated at transcription level by different bacterial pathogens. (C) 2010 Elsevier Ltd. All rights reserved.

Effects of increased pCO(2) and temperature on the North Atlantic spring bloom. I. The phytoplankton community and biogeochemical response

The North Atlantic spring bloom is one of the largest annual biological events in the ocean, and is characterized by dominance transitions from siliceous (diatoms) to calcareous (coccolithophores) algal groups. To study the effects of future global change on these phytoplankton and the biogeochemical cycles they mediate, a shipboard continuous culture experiment (Ecostat) was conducted in June 2005 during this transition period. Four treatments were examined: (1) 12 degrees C and 390 ppm CO2 (ambient control), (2) 12 degrees C and 690 ppm CO2 (high pCO(2)) (3) 16 degrees C and 390 ppm CO2 (high temperature), and (4) 16 degrees C and 690 ppm CO2 ('greenhouse'). Nutrient availability in all treatments was designed to reproduce the low silicate conditions typical of this late stage of the bloom. Both elevated pCO(2) and temperature resulted in changes in phytoplankton community structure. Increased temperature promoted whole community photosynthesis and particulate organic carbon (POC) production rates per unit chlorophyll a. Despite much higher coccolithophore abundance in the greenhouse treatment, particulate inorganic carbon production (calcification) was significantly decreased by the combination of increased pCO(2) and temperature. Our experiments suggest that future trends during the bloom could include greatly reduced export of calcium carbonate relative to POC, thus providing a potential negative feedback to atmospheric CO2 concentration. Other trends with potential climate feedback effects include decreased community biogenic silica to POC ratios at higher temperature. These shipboard experiments suggest the need to examine whether future pCO2 and temperature increases on longer decadal timescales will similarly alter the biological and biogeochemical dynamics of the North Atlantic spring bloom.

Biochemical compositions of two dominant bloom- forming species isolated from the Yangtze River Estuary in response to different nutrient conditions

Variations of cellular total lipid, total carbohydrate and total protein content of two dominant bloom-forming species (Skeletonema costatum and Prorocentrum donghaiense) isolated from the Yangtze River Estuary were examined under six different nutrient conditions in batch cultures. Daily samples were collected to estimate the cell growth, nutrient concentration and three biochemical compositions content during 7 days for S. costatum and the same sampling procedure was done every other day during 10 days for P. donghaiense. Results showed that for S. costatum, cellular total lipid content increased under phosphorus (P) limitation, but not for nitrogen (N) limitation; cellular carbohydrate were accumulated under both N and P limitation: cellular total protein content of low nutrient concentration treatments were significantly lower than that of high nutrient concentration treatments. For P. donghaiense, both cellular total lipid content and total carbohydrate content were greatly elevated as a result of N and P exhaustion, but cellular total protein content had no significant changes under nutrient limitation. In addition, the capability of accumulation of three biochemical constituents of P. donghaiense was much stronger than that of S. costatum. Pearson correlation showed that for both species, the biochemical composition of three constituents (lipid, carbohydrate and protein) had no significant relationship with extracellular N concentration, but had positive correlation with extracellular and intracellular P concentration. The capability of two species to accumulate cellular total lipid and carbohydrate under nutrient limitation may help them accommodate the fluctuating nutrient condition of the Yangtze River Estuary. The different responses of two species of cellular biochemical compositions content under different nutrient conditions may provide some evidence to explain the temporal characteristic of blooms Caused by two species in the Yangtze River Estuary. (C) 2008 Elsevier B.V. All rights reserved.

Response of cyanobacterial carbon concentrating system to light intensity: a simulated analysis

Cyanobacteria possess a delicate system known as the carbon concentrating mechanism (CCM), which can efficiently elevate the intracellular inorganic carbon (Ci) concentration via active transportation. The system requires energy supplied by photosystems; therefore, the activity of the Ci transporter is closely related to light intensity. However, the relationship between CCM and light intensity has rarely been evaluated. Here, we present an improved quantitative model of CCM in which light is incorporated, and developed a CCM model that modified after Fridlyand et al. in 1996. Some equations used in this model were inducted to describe the relationship between transport capacity and light intensity, by which the response of the CCM to light change is simulated. Our results indicate that the efficiency of the carbon concentrating system is sensitive to light intensity. When the external Ci concentration was low, CO2 uptake dominated the total Ci uptake with increasing light intensity, while under high external Ci concentrations HCO3- uptake primarily contributed to the total Ci uptake. Variations in the ratio of energy allocated between the transport systems could markedly affect the operation of CCM. Indeed, our simulations suggest that various combinations of Ci fluxes can provide a possible approach to detect the way by which the cell distributes energy produced by the photosystems to the two active Ci transport processes. The proportion of the energy consumed on CCM to the total energy expenditure for the fixation of one CO2 molecule was determined at 18%-40%.

Intracellular copper/zinc superoxide dismutase from bay scallop Argopecten irradians: Its gene structure, mRNA expression and recombinant protein

Superoxide dismutases are an ubiquitous family of enzymes that function to efficiently catalyze the dismutation of superoxide anions. Two unique and highly compartmentalized bay scallop Argopecten irradians superoxide dismutases: MnSOD and ecCuZnSOD, have been molecularly characterized in our previous study. To complete characterize the SOD family in A. irradians, a novel intracellular copper/zinc SOD from the A. irradians (Ai-icCuZnSOD) was obtained and characterized. The full-length cDNA of Ai-icCuZnSOD was 1047 bp with a 459 bp open reading frame encoding 152 amino acids. The genomic length of the Ai-icCuZnSOD gene was about 4279 bp containing 4 exons and 3 introns. The promoter region containing many putative transcription factor binding sites were analyzed. Furthermore, quantitative reverse transcriptase real-time PCR (qRT-PCR) analysis indicated that the highest expression of the Ai-icCuZnSOD was detected in gill and the expression profiles in hemocytes of bay scallops challenged with bacteria Vibrio anguillarum and lipopolysaccharide (LPS) were different. The result presented an increased expression after injection with LPS whereas no significant changes were observed after V. anguillarum injection. A fusion protein containing Ai-icCuZnSOD was produced in vitro. The rAi-icCuZnSOD is a stable enzyme, retaining more than 80% of its activity between 10 and 60 degrees C and keeping above 88% of its activity at pH values between 5.8 and 9. Ai-icCuZnSOD is more stable under alkaline than acidic conditions. Crown Copyright (C) 2009 Published by Elsevier Ltd. All rights reserved.

Ryanodine receptor expression in trophoblasts

Trophoblasts of the placenta are the frontline cells involved in communication and exchange of materials between the mother and fetus. Within trophoblasts, calcium signalling proteins are richly expressed. Intracellular free calcium ions are a key second messenger, regulating various cellular activities. Transcellular Ca2+ transport through trophoblasts is essential in fetal skeleton formation. Ryanodine receptors (RyRs) are high conductance cation channels that mediate Ca2+ release from intracellular stores to the cytoplasm. To date, the roles of RyRs in trophoblasts have not been reported. By use of reverse transcription PCR and western blotting, the current study revealed that RyRs are expressed in model trophoblast cell lines (BeWo and JEG-3) and in human first trimester and term placental villi. Immunohistochemistry of human placental sections indicated that both syncytiotrophoblast and cytotrophoblast cell layers were positively stained by antibodies recognising RyRs; likewise, expression of RyR isoforms was also revealed in BeWo and JEG-3 cells by immunofluorescence microscopy. In addition, changes in [Ca2+]i were observed in both BeWo and JEG-3 cells upon application of various RyR agonists and antagonists, using fura-2 fluorescent videomicroscopy. Furthermore, endogenous placental peptide hormones, namely angiotensin II, arginine vasopressin and endothelin 1, were demonstrated to increase [Ca2+]i in BeWo cells, and such increases were suppressed by RyR antagonists and by blockers of the corresponding peptide hormone receptors. These findings indicate that 1) multiple RyR subtypes are expressed in human trophoblasts; 2) functional RyRs in BeWo and JEG-3 cells response to both RyR agonists and antagonists; 3) RyRs in BeWo cells mediate Ca2+ release from intracellular store in response to the indirect stimulation by endogenous peptides. These observations suggest that RyR contributes to trophoblastic cellular Ca2+ homeostasis; trophoblastic RyRs are also involved in the functional regulation of human placenta by coupling to endogenous placental peptide-induced signalling pathways.