Shifts in the relative availability of phosphorus and nitrogen along estuarine salinity gradients

Phosphorus (P) availability in estuaries may increase with increasing salinity because sulfate from sea salt supports production of sulfide in sediments, which combines with iron (Fe) making it less available to sequester P. Increased P availability with increased salinity may promote the generally observed switch from P limitation of primary production in freshwater ecosystems to nitrogen (N) limitation in coastal marine waters. To investigate this hypothesis, we analyzed pore water from sediment cores collected along the salinity gradients of four Chesapeake Bay estuaries (the Patuxent, Potomac, Choptank, and Bush Rivers) with watersheds differing in land cover and physiography. At salinities of 1–4 in each estuary, abrupt decreases in pore water Fe²⁺ concentrations coincided with increases in sulfate depletion and PO₄ ^3? concentrations. Peaks in water column PO₄ ^3? concentrations also occur at about the same position along the salinity gradient of each estuary. Increases in pore water PO₄ ^3? concentration with increasing salinity led to distinct shifts in molar NH₄ ⁺:PO₄ ^3? ratios from >16 (the Redfield ratio characteristic of phytoplankton N:P) in the freshwater cores to <16 in the cores with salinities >1 to 4, suggesting that release of PO₄ ^3? from Fe where sediments are first deposited in sulfate-rich waters could promote the commonly observed switch from P limitation in freshwater to N limitation in mesohaline waters. Finding this pattern at similar salinities in four estuaries with such different watersheds suggests that it may be a fundamental characteristic of estuaries generally.     

Bestrophin Cl- channels are highly permeable to HCO3-

Bestrophin-1 (Best1) is a Cl(-) channel that is linked to various retinopathies in both humans and dogs. Dysfunction of the Best1 Cl(-) channel has been proposed to cause retinopathy because of altered Cl(-) transport across the retinal pigment epithelium (RPE). In addition to Cl(-), many Cl(-) channels also transport HCO3(-). Because HCO3(-) is physiologically important in pH regulation and in fluid and ion transport across the RPE, we measured the permeability and conductance of bestrophins to HCO3(-) relative to Cl(-). Four human bestrophin homologs (hBest1, hBest2, hBest3, and hBest4) and mouse Best2 (mBest2) were expressed in HEK cells, and the relative HCO3(-) permeability (P HCO3/PCl) and conductance (G HCO3/GCl) were determined. P HCO3/PCl was calculated from the change in reversal potential (Erev) produced by replacing extracellular Cl(-) with HCO3(-). hBest1 was highly permeable to HCO3(-) (P HCO3)/PCl = approximately 0.44). hBest2, hBest4, and mBest2 had an even higher relative HCO3(-) permeability (P HCO3/PCl = 0.6-0.7). All four bestrophins had HCO3(-) conductances that were nearly the same as Cl(-) (G HCO3/GCl = 0.9-1.1). Extracellular Na+ did not affect the permeation of hBest1 to HCO3(-). At physiological HCO3(-) concentration, HCO3(-) was also highly conductive. The hBest1 disease-causing mutations Y85H, R92C, and W93C abolished both Cl(-) and HCO3(-) currents equally. The V78C mutation changed P HCO3/PCl and G HCO3/GCl of mBest2 channels. These results raise the possibility that disease-causing mutations in hBest1 produce disease by altering HCO3(-) homeostasis as well as Cl(-) transport in the retina.     

Temperate Myxococcus xanthus phage Mx8 encodes a DNA adenine methylase, Mox.

Temperate bacteriophage Mx8 of Myxococcus xanthus encapsidates terminally repetitious DNA, packaged as circular permutations of its 49-kbp genome. During both lytic and lysogenic development, Mx8 expresses a nonessential DNA methylase, Mox, which modifies adenine residues in occurrences of XhoI and PstI recognition sites, CTCGAG and CTGCAG, respectively, on both phage DNA and the host chromosome. The mox gene is necessary for methylase activity in vivo, because an amber mutation in the mox gene abolishes activity. The mox gene is the only phage gene required for methylase activity in vivo, because ectopic expression of mox as part of the M. xanthus mglBA operon results in partial methylation of the host chromosome. The predicted amino acid sequence of Mox is related most closely to that of the methylase involved in the cell cycle control of Caulobacter crescentus. We speculate that Mox acts to protect Mx8 phage DNA against restriction upon infection of a subset of natural M. xanthus hosts. One natural isolate of M. xanthus, the lysogenic source of related phage Mx81, produces a restriction endonuclease with the cleavage specificity of endonuclease BstBI.     

Acetycholine receptor production and incorporation into membranes of developing muscle fibers

Using electrophysiological and quantitative autoradiographic techniques, we studied the kinetics of acetylcholine (ACh) receptor production and incorporation into membranes of muscle fibers developing in culture. These studies were performed by utilizing ¹²⁵I-labeled α-Bungarotoxin (α-BGT) which binds irreversibly to ACh receptors. α-BGT binding to ACh-sensitive muscle cells in culture correlates well with the level of ACh sensitivity. α-BGT binds to myotubes with two different apparent rates. The slow component of binding is due to the incorporation of new receptors into the membrane at a rate of 90 receptors/μm² per hour. However, the ACh receptor density increases at a rate of only 35 receptors/μm² per hour as the result of a concurrent increase in cell surface area. The α-BGT-receptor complexes turn over slowly and the rate of receptor incorporation is not affected by the presence of α-BGT. Inhibition of protein synthesis with cycloheximide depresses receptor incorporation, the percent inhibition increasing with time in cycloheximide. Overnight treatment in actinomycin D has no effect, but inhibition of ATP synthesis with dinitrophenol and iodoacetate or incubation in the cold inhibits the appearance of new ACh receptors.     

Dynamics of calcium regulation of chloride currents in Xenopus oocytes

Ca-activated Cl currents are widely expressed in many cell typesand play diverse and important physiological roles. TheXenopus oocyte is a good model systemfor studying the regulation of these currents. We previously showedthat inositol 1,4,5-trisphosphate (IP₃) injection intoXenopus oocytes rapidly elicits anoninactivating outward Cl current(I_Cl1-S)followed several minutes later by the development of slow inward(I_Cl2) andtransient outward(I_Cl1-T) Clcurrents. In this paper, we investigate whether these three currentsare mediated by the same or different Cl channels. Outward Cl currentswere more sensitive to Ca than inward Cl currents, as shown byinjection of different amounts of Ca or by Ca influx through aheterologously expressed ligand-gated Ca channel, the ionotropicglutamate receptor iGluR3. These data could be explained by twochannels with different Ca affinities or one channel with a higher Caaffinity at depolarized potentials. To distinguish between thesepossibilities, we determined the anion selectivity of the threecurrents. The anion selectivity sequences for the three currents werethe same (I > Br > Cl), butI_Cl1-Shad an I-to-Cl permeability ratio more than twofold smaller than the other two currents. The different anion selectivities and instantaneous current-voltage relationships were consistent with at least two different channels mediating these currents. However, afterconsideration of possible errors, the hypothesis that a single type ofCl channel underlies the complex waveforms of the three differentmacroscopic Ca-activated Cl currents inXenopus oocytes remains a viable alternative.

     

CNS melanocortin and leptin effects on stearoyl-CoA desaturase-1 and resistin expression

The objective of this study was to determine whether centrally administered leptin decreased liver and adipose SCD1 expression or adipose resistin expression, and whether these effects were mediated by central melanocortin receptors. Male Sprague-Dawley rats were injected into the lateral cerebral ventricle (i.c.v.) once daily for 4 days with artificial cerebrospinal fluid (aCSF, 5 microl), leptin (10 microg) or MTII (0.1 nmol); two other groups were pretreated icv with the melanocortin antagonist, SHU9119 (1.0 nmol), followed by leptin or MTII. Epididymal and inguinal adipose tissue and liver were collected after rats were killed and mRNA expression of SCD1 and resistin was measured. Both leptin and MTII reduced SCD1 expression and pretreatment with SHU9119 reversed their effects. Neither leptin nor MTII affected resistin expression, but it was increased by SHU9119. These results show that CNS melanocortin receptors are likely mediators of leptin's effects on SCD1 expression in liver and adipose tissue, The findings were inconclusive concerning the effects of leptin and melanocortins on adipose resistin expression.     

Characterization of the oligomeric structure of the Ca(2+)-activated Cl- channel Ano1/TMEM16A

Members of the Anoctamin (Ano)/TMEM16A family have recently been identified as essential subunits of the Ca²⁺-activated chloride channel (CaCC). For example, Ano1 is highly expressed in multiple tissues including airway epithelia, where it acts as an apical conduit for transepithelial Cl⁻ secretion and helps regulate lung liquid homeostasis and mucus clearance. However, little is known about the oligomerization of this protein in the plasma membrane. Thus, utilizing mCherry- and eGFP-tagged Ano1 constructs, we conducted biochemical and Förster resonance energy transfer (FRET)-based experiments to determine the quaternary structure of Ano1. FRET and co-immunoprecipitation studies revealed that tagged Ano1 subunits directly associated before they reached the plasma membrane. This association was not altered by changes in cytosolic Ca²⁺, suggesting that this is a fixed interaction. To determine the oligomeric structure of Ano1, we performed chemical cross-linking, non-denaturing PAGE, and electromobility shift assays, which revealed that Ano1 exists as a dimer. These data are the first to probe the quaternary structure of Ano1. Understanding the oligomeric nature of Ano1 is an essential step in the development of therapeutic drugs that could be useful in the treatment of cystic fibrosis.     

Potentiation of oxidative cell injury in hepatocytes which have accumulated Ca2+

Incubation of freshly isolated rat hepatocytes with exogenous ATP, but not with succinate, resulted in intracellular Ca2+ accumulation which was partly prevented when the inhibitor of mitochondrial Ca2+ sequestration, ruthenium red, was also present in the medium. Although the bulk of the accumulated Ca2+ was sequestered by the mitochondria, formation of surface blebs and stimulation of phosphorylase alpha activity during incubation of the hepatocytes with ATP indicate that this treatment was also associated with an increase in cytosolic free Ca2+ concentration. When hepatocytes loaded with Ca2+ by preincubation with ATP were exposed to either 2-methyl-1,4-naphthoquinone or t-butyl hydroperoxide, the cytotoxicity of both agents was markedly potentiated. Our results suggest that ATP-induced Ca2+ accumulation in hepatocytes is not due to contamination of the cell suspension with damaged cells or free intracellular organelles and that the intracellular Ca2+ concentration can affect the response to toxic agents.     

Functional geometry of the permeation pathway of Ca2+-activated Cl-channels inferred from analysis of voltage-dependent block

We examined the voltage-dependent block of Ca(2+)-activated Cl(-) channels by anthacene-9-carboxylic acid (A9C), diphenylamine-2-carboxylic acid (DPC), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and niflumic acid (NFA) in excised inside-out and outside-out patches from Xenopus oocytes. The fraction of the voltage field (delta) experienced by the blocking drug was determined from the voltage dependence of block. All the drugs blocked by entering the channel from the outside. delta was 0.6 for A9C, 0.3 for DPC and DIDS, and <0.1 for NFA. Because the voltage dependence of the drugs differed, the order of potency was also voltage-dependent. At +100 mV the order of potency was NFA > A9C > DIDS > DPC (K(i) (microm) = 10.1, 18.3, 48, and 111, respectively). Because the drugs are hydrophobic, they can cross the bilayer when applied from the inside and block the channel from the outside. The equilibrium geometries of the blockers were determined by molecular modeling and compared with their blocking positions (delta). This analysis suggests that the channel is an elliptical cone with the largest opening facing the extracellular space. The selectivity filter has an apparent size of 0.33 x 0.75 nm, because C(CN)(3)-, which has these dimensions, permeates. The external opening is at least 0.60 x 0.94 nm, because DPC has these dimensions and penetrates the channel approximately 30%.