Olan Jackson-Weaver

Molecular Characterization of the Diversity and Distribution of a Thermal Spring Microbial Community by Using rRNA and Metabolic Genes

ABSTRACT The diversity and distribution of a bacterial community from Coffee Pots Hot Spring, a thermal spring in Yellowstone National Park with a temperature range of 39.3 to 74.1°C and pH range of 5.75 to 6.91, were investigated by sequencing cloned PCR products and quantitative PCR (qPCR) of 16S rRNA and metabolic genes. The spring was inhabited by three Aquificae genera—Thermocrinis, Hydrogenobaculum, and Sulfurihydrogenibium—and members of the Alpha-, Beta-, and Gammaproteobacteria, Firmicutes, Acidobacteria, Deinococcus-Thermus, and candidate division OP5. The in situ chemical affinities were calculated for 41 potential metabolic reactions using measured environmental parameters and a range of hydrogen and oxygen concentrations. Reactions that use oxygen, ferric iron, sulfur, and nitrate as electron acceptors were predicted to be the most energetically favorable, while reactions using sulfate were expected to be less favorable. Samples were screened for genes used in ammonia oxidation (amoA, bacterial gene only), the reductive tricarboxylic acid (rTCA) cycle (aclB), the Calvin cycle (cbbM), sulfate reduction (dsrAB), nitrogen fixation (nifH), nitrite reduction (nirK), and sulfide oxidation (soxEF1) by PCR. Genes for carbon fixation by the rTCA cycle and nitrogen fixation were detected. All aclB sequences were phylogenetically related and spatially correlated to Sulfurihydrogenibium 16S rRNA gene sequences using qPCR (R2 = 0.99). This result supports the recent finding of citrate cleavage by enzymes other than ATP citrate lyase in the rTCA cycle of the Aquificaceae family. We briefly consider potential biochemical mechanisms that may allow Sulfurihydrogenibium and Thermocrinis to codominate some hydrothermal environments.

Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses

Intracellular Ca2+ transients are considered a primary signal by which astrocytes interact with neurons and blood vessels. With existing commonly used methods, Ca2+ has been studied only within astrocyte somata and thick branches, leaving the distal fine branchlets and endfeet that are most proximate to neuronal synapses and blood vessels largely unexplored. Here, using cytosolic and membrane-tethered forms of genetically encoded Ca2+ indicators (GECIs; cyto-GCaMP3 and Lck-GCaMP3), we report well-characterized approaches that overcome these limitations. We used in vivo microinjections of adeno-associated viruses to express GECIs in astrocytes and studied Ca2+ signals in acute hippocampal slices in vitro from adult mice (aged ∼P80) two weeks after infection. Our data reveal a sparkling panorama of unexpectedly numerous, frequent, equivalently scaled, and highly localized Ca2+ microdomains within entire astrocyte territories in situ within acute hippocampal slices, consistent with the distribution of perisynaptic branchlets described using electron microscopy. Signals from endfeet were revealed with particular clarity. The tools and experimental approaches we describe in detail allow for the systematic study of Ca2+ signals within entire astrocytes, including within fine perisynaptic branchlets and vessel-associated endfeet, permitting rigorous evaluation of how astrocytes contribute to brain function.

TRPA1 channels are regulators of astrocyte basal calcium levels and long-term potentiation via constitutive D-serine release.

Astrocytes are found throughout the brain where they make extensive contacts with neurons and synapses. Astrocytes are known to display intracellular Ca2+ signals and release signaling molecules such as d-serine into the extracellular space. However, the role(s) of astrocyte Ca2+ signals in hippocampal long-term potentiation (LTP), a form of synaptic plasticity involved in learning and memory, remains unclear. Here, we explored a recently discovered novel TRPA1 channel-mediated transmembrane Ca2+ flux pathway in astrocytes. Specifically, we determined whether block or genetic deletion of TRPA1 channels affected LTP of Schaffer collateral to CA1 pyramidal neuron synapses. Using pharmacology, TRPA1−/− mice, imaging, electrophysiology, and d-serine biosensors, our data indicate that astrocyte TRPA1 channels contribute to basal Ca2+ levels and are required for constitutive d-serine release into the extracellular space, which contributes to NMDA receptor-dependent LTP. The findings have broad relevance for the study of astrocyte–neuron interactions by demonstrating how TRPA1 channel-mediated fluxes contribute to astrocyte basal Ca2+ levels and neuronal function via constitutive d-serine release.

Conditions and constraints for astrocyte calcium signaling in the hippocampal mossy fiber pathway

The spatiotemporal activities of astrocyte Ca²⁺ signaling in mature neuronal circuits remain unclear. We used genetically encoded Ca²⁺ and glutamate indicators as well as pharmacogenetic and electrical control of neurotransmitter release to explore astrocyte activity in the hippocampal mossy fiber pathway. Our data revealed numerous localized, spontaneous Ca²⁺ signals in astrocyte branches and territories, but these were not driven by neuronal activity or glutamate. Moreover, evoked astrocyte Ca²⁺ signaling changed linearly with the number of mossy fiber action potentials. Under these settings, astrocyte responses were global, suppressed by neurotransmitter clearance, and mediated by glutamate and GABA. Thus, astrocyte engagement in the fully developed mossy fiber pathway was slow and territorial, contrary to that frequently proposed for astrocytes within microcircuits. We show that astrocyte Ca²⁺ signaling functionally segregates large volumes of neuropil and that these transients are not suited for responding to, or regulating, single synapses in the mossy fiber pathway.

Intermittent Hypoxia in Rats Increases Myogenic Tone Through Loss of Hydrogen Sulfide Activation of Large-Conductance Ca 2+ -Activated Potassium Channels

Rationale: Myogenic tone, an important regulator of vascular resistance, is dependent on vascular smooth muscle (VSM) depolarization, can be modulated by endothelial factors, and is increased in several models of hypertension. Intermittent hypoxia (IH) elevates blood pressure and causes endothelial dysfunction. Hydrogen sulfide (H2S), a recently described endothelium-derived vasodilator, is produced by the enzyme cystathionine &ggr;-lyase (CSE) and acts by hyperpolarizing VSM. Objective: Determine whether IH decreases endothelial H2S production to increase myogenic tone in small mesenteric arteries. Methods and Results: Myogenic tone was greater in mesenteric arteries from IH than sham control rat arteries, and VSM membrane potential was depolarized in IH in comparison with sham arteries. Endothelium inactivation or scavenging of H2S enhanced myogenic tone in sham arteries to the level of IH. Inhibiting CSE also enhanced myogenic tone and depolarized VSM in sham but not IH arteries. Similar results were seen in cerebral arteries. Exogenous H2S dilated and hyperpolarized sham and IH arteries, and this dilation was blocked by iberiotoxin, paxilline, and KCl preconstriction but not glibenclamide or 3-isobutyl-1-methylxanthine. Iberiotoxin enhanced myogenic tone in both groups but more in sham than IH. CSE immunofluorescence was less in the endothelium of IH than in sham mesenteric arteries. Endogenouse H2S dilation was reduced in IH arteries. Conclusions: IH appears to decrease endothelial CSE expression to reduce H2S production, depolarize VSM, and enhance myogenic tone. H2S dilatation and hyperpolarization of VSM in small mesenteric arteries requires BKCa channels.

Hydrogen sulfide dilates rat mesenteric arteries by activating endothelial large-conductance Ca2+-activated K+ channels and smooth muscle Ca2+ sparks

We have previously shown that hydrogen sulfide (H₂S) reduces myogenic tone and causes relaxation of phenylephrine (PE)-constricted mesenteric arteries. This effect of H₂S to cause vasodilation and vascular smooth muscle cell (VSMC) hyperpolarization was mediated by large-conductance Ca(2+)-activated potassium channels (BKCa). Ca(2+) sparks are ryanodine receptor (RyR)-mediated Ca(2+)-release events that activate BKCa channels in VSMCs to cause membrane hyperpolarization and vasodilation. We hypothesized that H₂S activates Ca(2+) sparks in small mesenteric arteries. Ca(2+) sparks were measured using confocal microscopy in rat mesenteric arteries loaded with the Ca(2+) indicator fluo-4. VSMC membrane potential (Em) was measured in isolated arteries using sharp microelectrodes. In PE-constricted arteries, the H₂S donor NaHS caused vasodilation that was inhibited by ryanodine (RyR blocker), abluminal or luminal iberiotoxin (IbTx, BKCa blocker), endothelial cell (EC) disruption, and sulfaphenazole [cytochrome P-450 2C (Cyp2C) inhibitor]. The H₂S donor NaHS (10 μmol/l) increased Ca(2+) sparks but only in the presence of intact EC and this was blocked by sulfaphenazole or luminal IbTx. Inhibiting cystathionine γ-lyase (CSE)-derived H2S with β-cyano-l-alanine (BCA) also reduced VSMC Ca(2+) spark frequency in mesenteric arteries, as did EC disruption. However, excess CSE substrate homocysteine did not affect spark activity. NaHS hyperpolarized VSMC Em in PE-depolarized mesenteric arteries with intact EC and also hyperpolarized EC Em in arteries cut open to expose the lumen. This hyperpolarization was prevented by ryanodine, sulfaphenazole, and abluminal or luminal IbTx. BCA reduced IbTx-sensitive K(+) currents in freshly dispersed mesenteric ECs. These results suggest that H₂S increases Ca(2+) spark activity in mesenteric artery VSMC through activation of endothelial BKCa channels and Cyp2C, a novel vasodilatory pathway for this emerging signaling molecule.

Mechanisms of diesel-induced endothelial nitric oxide synthase dysfunction in coronary arterioles.

Background and objective Increased air pollutants correlate with increased incidence of cardiovascular disease potentially due to vascular dysfunction. We have reported that acute diesel engine exhaust (DE) exposure enhances vasoconstriction and diminishes acetylcholine (ACh)-induced dilation in coronary arteries in a nitric oxide synthase (NOS)-dependent manner. We hypothesize that acute DE inhalation leads to endothelial dysfunction by uncoupling NOS. Methods Rats inhaled fresh DE (300 μg particulate matter/m3) or filtered air for 5 hr. After off-gassing, intraseptal coronary arteries were isolated and dilation to ACh recorded using videomicroscopy. Results Arteries from DE-exposed animals dilated less to ACh than arteries from air-exposed animals. NOS inhibition did not affect ACh dilation in control arteries but increased dilation in the DE group, suggesting NOS does not normally contribute to ACh-induced dilation in coronary arteries but does contribute to endothelial dysfunction after DE inhalation. Cyclooxygenase (COX) inhibition did not affect ACh dilation in the DE group, but combined inhibition of NOS and COX diminished dilation in both groups and eliminated intergroup differences, suggesting that the two pathways interact. Superoxide scavenging increased ACh dilation in DE arteries, eliminating differences between groups. Tetrahydrobiopterin (BH4) supplementation with sepiapterin restored ACh-mediated dilation in the DE group in a NOS-dependent manner. Superoxide generation (dihydroethidium staining) was greater in DE arteries, and superoxide scavenging, BH4 supplementation, or NOS inhibition reduced the signal in DE but not air arteries. Conclusion Acute DE exposure appears to uncouple NOS, increasing reactive oxygen species generation and causing endothelial dysfunction, potentially because of depletion of BH4 limiting its bioavailability.

Volcanic calderas delineate biogeographic provinces among Yellowstone thermophiles

It has been suggested that the distribution of microorganisms should be cosmopolitan because of their enormous capacity for dispersal. However, recent studies have revealed that geographically isolated microbial populations do exist. Geographic distance as a barrier to dispersal is most often invoked to explain these distributions. Here we show that unique and diverse sequences of the bacterial genus Sulfurihydrogenibium exist in Yellowstone thermal springs, indicating that these sites are geographically isolated. Although there was no correlation with geographic distance or the associated geochemistry of the springs, there was a strong historical signal. We found that the Yellowstone calderas, remnants of prehistoric volcanic eruptions, delineate biogeographical provinces for the Sulfurihydrogenibium within Yellowstone (chi(2): 9.7, P = 0.002). The pattern of distribution that we have detected suggests that major geological events in the past 2 million years explain more of the variation in sequence diversity in this system than do contemporary factors such as habitat or geographic distance. These findings highlight the importance of historical legacies in determining contemporary microbial distributions and suggest that the same factors that determine the biogeography of macroorganisms are also evident among bacteria.

NFATc3 is required for intermittent hypoxia-induced hypertension

Sleep apnea, defined as intermittent respiratory arrest during sleep, is associated with increased incidence of hypertension and peripheral vascular disease. Exposure of rodents to brief periods of intermittent hypercarbia/hypoxia (H-IH) during sleep mimics the cyclical hypoxia-normoxia of sleep apnea. Endothelin-1, an upstream activator of nuclear factor of activated T cells (NFAT), is increased during H-IH. Therefore, we hypothesized that NFATc3 is activated by H-IH and is required for H-IH-induced hypertension. Consistent with this hypothesis, we found that H-IH (20 brief exposures per hour to 5% O(2)-5% CO(2) for 7 h/day) induces systemic hypertension in mice [mean arterial pressure (MAP) = 97 +/- 2 vs. 124 +/- 2 mmHg, P < 0.05, n = 5] and increases NFATc3 transcriptional activity in aorta and mesenteric arteries. Cyclosporin A, an NFAT inhibitor, and genetic ablation of NFATc3 [NFATc3 knockout (KO)] prevented NFAT activation. More importantly, H-IH-induced hypertension was attenuated in cyclosporin A-treated mice and prevented in NFATc3 KO mice. MAP was significantly elevated in wild-type mice (Delta = 23.5 +/- 6.1 mmHg), but not in KO mice (Delta = -3.9 +/- 5.7). These results indicate that H-IH-induced increases in MAP require NFATc3 and that NFATc3 may contribute to the vascular changes associated with H-IH-induced hypertension.

Phosphoinositide-Dependent Kinase-1 and Protein Kinase Cδ Contribute to Endothelin-1 Constriction and Elevated Blood Pressure in Intermittent Hypoxia

Obstructive sleep apnea (OSA) is associated with cardiovascular complications including hypertension. Previous findings from our laboratory indicate that exposure to intermittent hypoxia (IH), to mimic sleep apnea, increases blood pressure in rats. IH also increases endothelin-1 (ET-1) constrictor sensitivity in a protein kinase C (PKC) δ–dependent manner in mesenteric arteries. Because phosphoinositide-dependent kinase-1 (PDK-1) regulates PKCδ activity, we hypothesized that PDK-1 contributes to the augmented ET-1 constrictor sensitivity and elevated blood pressure following IH. Male Sprague-Dawley rats were exposed to either sham or IH (cycles between 21% O2/0% CO2 and 5% O2/5% CO2) conditions for 7 h/day for 14 or 21 days. The contribution of PKCδ and PDK-1 to ET-1–mediated vasoconstriction was assessed in mesenteric arteries using pharmacological inhibitors. Constrictor sensitivity to ET-1 was enhanced in arteries from IH-exposed rats. Inhibition of PKCδ or PDK-1 blunted ET-1 constriction in arteries from IH but not sham group rats. Western analysis revealed similar levels of total and phosphorylated PDK-1 in arteries from sham and IH group rats but decreased protein-protein interaction between PKCδ and PDK-1 in arteries from IH- compared with sham-exposed rats. Blood pressure was increased in rats exposed to IH, and treatment with the PDK-1 inhibitor OSU-03012 [2-amino-N-{4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-phenyl}-acetamide] (33 mg/day) lowered blood pressure in IH but not sham group rats. Our results suggest that exposure to IH unmasks a role for PDK-1 in regulating ET-1 constrictor sensitivity and blood pressure that is not present under normal conditions. These novel findings suggest that PDK-1 may be a uniquely effective antihypertensive therapy for OSA patients.