Short-Term Regulation of Nitrogenase Activity by NH4+ in Rhodobacter capsulatus: Multiple In Vivo Nitrogenase Responses to NH4+ Addition

The photosynthetic bacterium Rhodobacter capsulatus has been shown to carry out nitrogenase “switch-off,” a rapid, reversible inhibition of in vivo activity. Here, we demonstrate that highly nitrogen-limited cultures of both the wild-type strain and a draT draG mutant are capable of nitrogenase switch-off while moderately nitrogen-limited cultures show instead a “magnitude” response, with a decrease in in vivo nitrogenase activity that is proportional to the amount of added NH₄⁺.     

Low-Concentration Kinetics of Atmospheric CH4 Oxidation in Soil and Mechanism of NH4+ Inhibition

NH₄⁺ inhibition kinetics for CH₄ oxidation were examined at near-atmospheric CH₄ concentrations in three upland forest soils. Whether NH₄⁺-independent salt effects could be neutralized by adding nonammoniacal salts to control samples in lieu of deionized water was also investigated. Because the levels of exchangeable endogenous NH₄⁺ were very low in the three soils, desorption of endogenous NH₄⁺ was not a significant factor in this study. The K_m(app) values for water-treated controls were 9.8, 22, and 57 nM for temperate pine, temperate hardwood, and birch taiga soils, respectively. At CH₄ concentrations of ≤15 μl liter⁻¹, oxidation followed first-order kinetics in the fine-textured taiga soil, whereas the coarse-textured temperate soils exhibited Michaelis-Menten kinetics. Compared to water controls, the K_m(app) values in the temperate soils increased in the presence of NH₄⁺ salts, whereas the V_max(app) values decreased substantially, indicating that there was a mixture of competitive and noncompetitive inhibition mechanisms for whole NH₄⁺ salts. Compared to the corresponding K⁺ salt controls, the K_m(app) values for NH₄⁺ salts increased substantially, whereas the V_max(app) values remained virtually unchanged, indicating that NH₄⁺ acted by competitive inhibition. Nonammoniacal salts caused inhibition to increase with increasing CH₄ concentrations in all three soils. In the birch taiga soil, this trend occurred with both NH₄⁺ and K⁺ salts, and the slope of the increase was not affected by the addition of NH₄⁺. Hence, the increase in inhibition resulted from an NH₄⁺-independent mechanism. These results show that NH₄⁺ inhibition of atmospheric CH₄ oxidation resulted from enzymatic substrate competition and that additional inhibition that was not competitive resulted from a general salt effect that was independent of NH₄⁺.     

CD4+-T-Cell and CD20+-B-Cell Changes Predict Rapid Disease Progression after Simian-Human Immunodeficiency Virus Infection in Macaques

Simian-human immunodeficiency virus 89.6PD (SHIV_89.6PD) was pathogenic after intrarectal inoculation of rhesus macaques. Infection was achieved with a minimum of 2,500 tissue culture infectious doses of cell-free virus stock, and there was no evidence for transient viremia in animals receiving subinfectious doses by the intrarectal route. Some animals experienced rapid progression of disease characterized by loss of greater than 90% of circulating CD4⁺ T cells, sustained decreases in CD20⁺ B cells, failure to elicit virus-binding antibodies in plasma, and high levels of antigenemia. Slower-progressing animals had moderate but varying losses of CD4⁺ T cells; showed increases in circulating CD20⁺ B cells; mounted vigorous responses to antibodies in plasma, including neutralizing antibodies; and had low or undetectable levels of antigenemia. Rapid progression led to death within 30 weeks after intrarectal inoculation. Plasma antigenemia at 2 weeks after inoculation (P ≤ 0.002), B- and T-cell losses (P ≤ 0.013), and failure to seroconvert (P ≤ 0.005) were correlated statistically with rapid progression. Correlations were evident by 2 to 4 weeks after intrarectal SHIV inoculation, indicating that early events in the host-pathogen interaction determined the clinical outcome.     

Altered Na+ and Li+ Homeostasis in Saccharomyces cerevisiae Cells Expressing the Bacterial Cation Antiporter NhaA

The bacterial Na⁺(Li⁺)/H⁺ antiporter NhaA has been expressed in the yeast Saccharomyces cerevisiae. NhaA was present in both the plasma membrane and internal membranes, and it conferred lithium but not sodium tolerance. In cells containing the yeast Ena1-4 (Na⁺, Li⁺) extrusion ATPase, the extra lithium tolerance conferred by NhaA was dependent on a functional vacuolar H⁺ ATPase and correlated with an increase of lithium in an intracellular pool which exhibited slow efflux of cations. In yeast mutants without (Na⁺, Li⁺) ATPase, lithium tolerance conferred by NhaA was not dependent on a functional vacuolar H⁺ ATPase and correlated with a decrease of intracellular lithium. NhaA was able to confer sodium tolerance and to decrease intracellular sodium accumulation in a double mutant devoid of both plasma membrane (Na⁺, Li⁺) ATPase and vacuolar H⁺ ATPase. These results indicate that the bacterial antiporter NhaA expressed in yeast is functional at both the plasma membrane and the vacuolar membrane. The phenotypes conferred by its expression depend on the functionality of plasma membrane (Na⁺, Li⁺) ATPase and vacuolar H⁺ ATPase.     

Defective Signaling in a Subpopulation of CD4+ T Cells in the Absence of Ca2+/Calmodulin-Dependent Protein Kinase IV

Ca(2+)/calmodulin-dependent protein kinase IV-deficient (CaMKIV(-/-)) mice have been used to investigate the role of this enzyme in CD4(+) T cells. We identify a functional defect in a subpopulation of CD4(+) T cells, characterized by a cell surface marker profile usually found on memory phenotype CD4(+) T cells. Upon T-cell receptor engagement, the mutant cells produce diminished levels of interleukin-2 (IL-2), IL-4, and gamma interferon protein and mRNA. The defect is secondary to an inability to phosphorylate CREB and to induce CREB-dependent immediate-early genes, including c-jun, fosB, fra2, and junB, which are required for cytokine gene induction. In contrast, stimulated naive CD4(+) T cells from CaMKIV(-/-) mice show normal CREB phosphorylation, induction of immediate-early genes, and cytokine production. Thus, in addition to defining an important signaling role for CaMKIV in a subpopulation of T cells, we identify differential signaling requirements for cytokine production between naive T cells and T cells that express cell surface markers characteristic of the memory phenotype.     

Polymorphism of Ca2+ Sparks Evoked from In-Focus Ca2+ Release Units in Cardiac Myocytes

Ca²⁺ sparks are the elementary release events in many types of cells. Here we present a morphometric analysis of Ca²⁺ sparks (i.e., amplitude and kinetic parameters) using an approach that minimizes the confounding factor of the detection of out-of-focus events. By activation and visualization of Ca²⁺ sparks from Ca²⁺ release units under loose-seal patch-clamp conditions, we found that the amplitude and rising rate of in-focus sparks exhibited a broad modal distribution, whereas spark rise time and spatial width appeared to be stereotyped. Spark morphometrics were constant irrespective of the latency of spark production and the time-dependent L-type Ca²⁺ channel activation. Polymorphism of Ca²⁺ sparks in terms of variable amplitude and rising rate was evident for events from the same release units, and intra- and interrelease unit variability contributed equally to the overall variability. The rising rate, a reporter of the underlying Ca²⁺ release flux, displayed a strong positive correlation with spark amplitude, but a negative correlation with spark rise time, an index of Ca²⁺ release duration. On the basis of Ca²⁺ spark morphometrics measured here, we suggested a model in which cohorts of variable number of ryanodine receptors are activated in the genesis of Ca²⁺ sparks, and the ensuing negative feedback overrides the regenerative Ca²⁺-induced Ca²⁺ release to extinguish the ongoing Ca²⁺ spark.     

Modulation of the Local SR Ca2+ Release by Intracellular Mg2+ in Cardiac Myocytes

In cardiac muscle, Ca²⁺-induced Ca²⁺ release (CICR) from the sarcoplasmic reticulum (SR) defines the amplitude and time course of the Ca²⁺ transient. The global elevation of the intracellular Ca²⁺ concentration arises from the spatial and temporal summation of elementary Ca²⁺ release events, Ca²⁺ sparks. Ca²⁺ sparks represent the concerted opening of a group of ryanodine receptors (RYRs), which are under the control of several modulatory proteins and diffusible cytoplasmic factors (e.g., Ca²⁺, Mg²⁺, and ATP). Here, we examined by which mechanism the free intracellular Mg²⁺ ([Mg²⁺]_free) affects various Ca²⁺ spark parameters in permeabilized mouse ventricular myocytes, such as spark frequency, duration, rise time, and full width, at half magnitude and half maximal duration. Varying the levels of free ATP and Mg²⁺ in specifically designed solutions allowed us to separate the inhibition of RYRs by Mg²⁺ from the possible activation by ATP and Mg²⁺-ATP via the adenine binding site of the channel. Changes in [Mg²⁺]_free generally led to biphasic alterations of the Ca²⁺ spark frequency. For example, lowering [Mg²⁺]_free resulted in an abrupt increase of spark frequency, which slowly recovered toward the initial level, presumably as a result of SR Ca²⁺ depletion. Fitting the Ca²⁺ spark inhibition by [Mg²⁺]_free with a Hill equation revealed a K_i of 0.1 mM. In conclusion, our results support the notion that local Ca²⁺ release and Ca²⁺ sparks are modulated by Mg²⁺ in the intracellular environment. This seems to occur predominantly by hindering Ca²⁺-dependent activation of the RYRs through competitive Mg²⁺ occupancy of the high-affinity activation site of the channels. These findings help to characterize CICR in cardiac muscle under normal and pathological conditions, where the levels of Mg²⁺ and ATP can change.     

Critical Role for CD4+ T Cells in Controlling Retrovirus Replication and Spread in Persistently Infected Mice

Reactivations of persistent viral infections pose a significant medical problem in immunocompromised cancer, transplant, and AIDS patients, yet little is known about how persistent viral infections are immunologically controlled. Here we describe a mouse model for investigating the role of the immune response in controlling a persistent retroviral infection. We demonstrate that, following recovery from acute Friend virus infection, a small number of B cells evade immunological destruction and harbor persistent virus. In vivo depletions of T-cell subsets in persistently infected mice revealed a critical role for CD4⁺ T cells in controlling virus replication, spread to the erythroid lineage, and induction of erythroleukemia. The CD4⁺ T-cell effect was independent of CD8⁺ T cells and in some cases was also independent of virus-neutralizing antibody responses. Thus, the CD4⁺ T cells may have had a direct antiviral effect. These results may have relevance for human immunodeficiency virus (HIV) infections where loss of CD4⁺ T cells is associated with an increase in HIV replication, reactivation of persistent viruses, and a high incidence of virus-associated cancers.     

Both Memory and CD45RA+/CD62L+ Naive CD4+ T Cells Are Infected in Human Immunodeficiency Virus Type 1-Infected Individuals

Cellular activation is critical for the propagation of human immunodeficiency virus type 1 (HIV-1) infection. It has been suggested that truly naive CD4(+) T cells are resistant to productive HIV-1 infection because of their constitutive resting state. Memory and naive CD4(+) T-cell subsets from 11 HIV-1-infected individuals were isolated ex vivo by a combination of magnetic bead depletion and fluorescence-activated cell sorting techniques with stringent criteria of combined expression of CD45RA and CD62L to identify naive CD4(+) T-cell subsets. In all patients HIV-1 provirus could be detected within naive CD45RA+/CD62L+ CD4(+) T cells; in addition, replication-competent HIV-1 was isolated from these cells upon CD4(+) T-cell stimulation in tissue cultures. Memory CD4(+) T cells had a median of fourfold more replication-competent virus and a median of sixfold more provirus than naive CD4(+) T cells. Overall, there was a median of 16-fold more integrated provirus identified in memory CD4(+) T cells than in naive CD4(+) T cells within a given patient. Interestingly, there was a trend toward equalization of viral loads in memory and naive CD4(+) T-cell subsets in those patients who harbored CXCR4-using (syncytium-inducing) viruses. Within any given patient, there was no selective usage of a particular coreceptor by virus isolated from memory versus naive CD4(+) T cells. Our findings suggest that naive CD4(+) T cells may be a significant viral reservoir for HIV, particularly in those patients harboring CXCR4-using viruses.     

Thymocyte-Thymic Epithelial Cell Interaction Leads to High-Level Replication of Human Immunodeficiency Virus Exclusively in Mature CD4+ CD8− CD3+ Thymocytes: a Critical Role for Tumor Necrosis Factor and Interleukin-7

This work aims at identifying the thymocyte subpopulation able to support human immunodeficiency virus (HIV) replication under the biological stimuli of the thymic microenvironment. In this report we demonstrate that interaction with thymic epithelial cells (TEC) induces a high-level replication of the T-tropic primary isolate HIV-1(B-LAIp) exclusively in the mature CD4(+) CD8(-) CD3(+) thymocytes. Tumor necrosis factor (TNF) and interleukin-7 (IL-7), secreted during this interaction, are critical cytokines for HIV long terminal repeat transactivation through NF-kappaB-dependent activation. TNF is the major inducer of NF-kappaB and particularly of the p50-p65 complex, whereas IL-7 acts as a cofactor by sustaining the expression of the p75 TNF receptor. The requirement for TNF is further confirmed by the observation that the inability of the intermediate CD4(+) CD8(-) CD3(-) thymocytes to replicate the virus is associated with a defect in TNF production during their interaction with TEC and correlates with the absence of nuclear NF-kappaB activity in these freshly isolated thymocytes. Addition of exogenous TNF to the intermediate thymocyte cultures induces NF-kappaB activity and is sufficient to promote HIV replication in the cocultures with TEC. The other major subpopulation expressing the CD4 receptor, namely, the double-positive (DP) CD4(+) CD8(+) CD3(+/-) thymocytes, despite the entry of the virus, do not produce a significant level of virus, presumably because they are unresponsive to TNF and IL-7. Together, these data suggest that in vivo, despite an efficient entry of the virus in all the CD4(+) subpopulations, a high viral load may be generated exclusively within the mature CD4(+) CD8(-) CD3(+) subset of thymocytes. However, under conditions of inflammatory response after infection, TNF might also be present in the intermediate thymocyte compartment, leading to efficient HIV replication in these cells.     

Phosphatidylinositol 3-Kinase-mediated Endocytosis of Renal Na+,K+-ATPase α Subunit in Response to Dopamine

Dopamine (DA) inhibition of Na⁺,K⁺-ATPase in proximal tubule cells is associated with increased endocytosis of its α and β subunits into early and late endosomes via a clathrin vesicle-dependent pathway. In this report we evaluated intracellular signals that could trigger this mechanism, specifically the role of phosphatidylinositol 3-kinase (PI 3-K), the activation of which initiates vesicular trafficking and targeting of proteins to specific cell compartments. DA stimulated PI 3-K activity in a time- and dose-dependent manner, and this effect was markedly blunted by wortmannin and LY 294002. Endocytosis of the Na⁺,K⁺-ATPase α subunit in response to DA was also inhibited in dose-dependent manner by wortmannin and LY 294002. Activation of PI 3-K generally occurs by association with tyrosine kinase receptors. However, in this study immunoprecipitation with a phosphotyrosine antibody did not reveal PI 3-K activity. DA-stimulated endocytosis of Na⁺,K⁺-ATPase α subunits required protein kinase C, and the ability of DA to stimulate PI 3-K was blocked by specific protein kinase C inhibitors. Activation of PI 3-K is mediated via the D₁ receptor subtype and the sequential activation of phospholipase A₂, arachidonic acid, and protein kinase C. The results indicate a key role for activation of PI 3-K in the endocytic sequence that leads to internalization of Na⁺,K⁺-ATPase α subunits in response to DA, and suggest a mechanism for the participation of protein kinase C in this process.     

Effects of Saxitoxin (STX) and Veratridine on Bacterial Na+-K+ Fluxes: a Prokaryote-Based STX Bioassay

Saxitoxin (STX) is a potent natural sodium channel blocker and represents a significant health concern worldwide. We describe here the antagonistic effects of STX and veratridine (VTD), an Na⁺ channel activator, on three gram-negative bacteria and their application to an STX bioassay. STX reduced the total cellular levels of both Na⁺ and K⁺, as measured by flame photometry, whereas VTD increased the cellular concentrations relative to control ion fluxes in the cyanobacterium Cylindrospermopsis raciborskii AWT205. Endogenous STX production in toxic cyanobacterial strains of C. raciborskii and Anabaena circinalis prevented cell lysis induced by VTD stress. Microscopic cell counts showed that non-STX producing cyanobacteria displayed complete cell lysis and trichome fragmentation 5 to 8 h after addition of VTD and vanadate (VAN), an inhibitor of sodium pumps. The addition of STX, or its analogue neoSTX, prior to treatment with VTD plus VAN prevented complete lysis in non-STX-producing cyanobacteria. VTD also affected cyanobacterial metabolism, and the presence of exogenous STX in the sample also ameliorated this decrease in metabolic activity, as measured by the cellular conversion of tetrazolium into formazan. Reduced primary metabolism was also recorded as a decrease in the light emissions of Vibrio fischeri exposed to VTD. Addition of STX prior to VTD resulted in a rapid and dose-dependent response to the presence of the channel blocker, with samples exhibiting resistance to the VTD effect. Our findings demonstrate that STX and VTD influence bacterial Na⁺ and K⁺ fluxes in opposite ways, and these principles can be applied to the development of a prokaryote-based STX bioassay.