Traditional epitranscriptomics relies on capturing a single RNA modification by antibody or chemical treatment, combined with short-read sequencing to identify its transcriptomic location. This approach is labor-intensive and may introduce experimental artifacts. Direct sequencing of native RNA using Oxford Nanopore Technologies (ONT) can allow for directly detecting the RNA base modifications, although these modifications might appear as sequencing errors. The percent Error of Specific Bases (%ESB) was higher for native RNA than unmodified RNA, which enabled the detection of ribonucleotide modification sites. Based on the %ESB differences, we developed a bioinformatic tool, epitranscriptional landscape inferring from glitches of ONT signals (ELIGOS), that is based on various types of synthetic modified RNA and applied to rRNA and mRNA. ELIGOS is able to accurately predict known classes of RNA methylation sites (AUC > 0.93) in rRNAs from Escherichiacoli, yeast, and human cells, using either unmodified in vitro transcription RNA or a background error model, which mimics the systematic error of direct RNA sequencing as the reference. The well-known DRACH/RRACH motif was localized and identified, consistent with previous studies, using differential analysis of ELIGOS to study the impact of RNA m6A methyltransferase by comparing wild type and knockouts in yeast and mouse cells. Lastly, the DRACH motif could also be identified in the mRNA of three human cell lines. The mRNA modification identified by ELIGOS is at the level of individual base resolution. In summary, we have developed a bioinformatic software package to uncover native RNA modifications.

Anion exchange plays an important role in renal ion transport and acidification. To further understand the molecular nature of renal epithelial anion exchange, we used a monoclonal antibody to the membrane domain (52 kDa) of human erythrocyte band 3 protein to immunocytochemically search for this polypeptide in the rabbit kidney. In cryostat sections, a subpopulation of cells in the cortical and outer medullary collecting tubules showed immunoreactivity; labeling was restricted to the basolateral membrane. Proximal tubules and thick and thin limbs of Henle showed no immunoreactivity. Approximately 11% of cells in the cortical, but 43% of cells in the medullary, collecting tubule were positive for band 3. To determine the type of cells that were band 3 positive, mitochondria-rich (intercalated) cells were identified by their positive histochemical staining for succinic dehydrogenase activity and by their ability to bind peanut lectin at the apical membrane. In the cortical collecting tubule, the majority of mitochondria-rich cells bound peanut lectin but were band 3 negative; the remainder were band 3 positive but lectin negative. This distribution was reversed in the inner stripe of the outer medulla: all mitochondria-rich cells were band 3 positive and lectin negative. Thus mitochondria-rich cells are of at least two types, each of which has a distinct axial distribution pattern. Given available information about in vitro HCO3 transport properties of rabbit collecting tubules, it is likely that the lectin-positive, band 3-negative mitochondria-rich cells secrete HCO3, whereas the lectin-negative, band 3-positive cells reabsorb HCO3 (secrete H).

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Red blood cells of several species are known to exhibit a ouabaininsensitive, anion-dependent K + (Rb § flux that is stimulated by cell swelling. We have used rabbit red cells to study the kinetics of activation and inactivation of the flux upon step changes in tonicity. Sudden hypotonic swelling (210 mosmol) activates the flux after a lag period of 10 min at 37"C and 30-50 min at 25"C. In cells that were preswollen to activate the transporter, sudden shrinkage (by addition of hypertonic NaC1) causes a rapid inactivation of the flux; the time lag for inactivation is <2 min at 37"C. A minimal model of the volume-sensitive KC1 transport system requires two states of the transporter. The activated (A) state catalyzes transport at some finite rate (turnover number unknown because the number of transporters is unknown). The resting (R) state has a much lower or possibly zero transport rate. The interconversion between the states is characterized by unimolecular rate constants R~A.k,21The rate of relaxation to any new steady state is equal to the sum of the rate constants k12 "{-k21. Because the rate of transport activation in a hypotonic medium is lower than the rate of inactivation in an isotonic medium, we conclude that the volume-sensitive rate process is inactivation (the A to R transition); that is, cell swelling activates transport by lowering k~l. Three phosphatase inhibitors (fluoride, orthovanadate, and inorganic phosphate) all inhibit the swelling-activated flux and also slow down the rate of approach to the swollen steady state. This finding suggests that a net dephosphorylation is necessary for activation of the flux and that the net dephosphorylation takes place as a result of swelling-induced inhibition of a kinase rather than stimulation of a phosphatase.

The mechanism of activation of KCl cotransport has been examined in rabbit red blood cells. Previous work has provided evidence that a net dephosphorylation is required for activation of transport by cell swelling. In the present study okadaic acid, an inhibitor of protein phosphatases, was used to test this idea in more detail. We find that okadaic acid strongly inhibits swelling-stimulated KCl cotransport. The IC50 for okadaic acid is approximately 40 nM, consistent with the involvement of type 1 protein phosphatase in transport activation. N-Ethylmaleimide (NEM) is well known to activate KCl cotransport in cells of normal volume. Okadaic acid, added before NEM, inhibits the activation of transport by NEM, indicating that a dephosphorylation is necessary for the NEM effect. Okadaic acid added after NEM inhibits transport only very slightly. After a brief exposure to NEM and rapid removal of unreacted NEM, KCl cotransport activates with a time delay that is similar to that for swelling activation. Okadaic acid causes a slight increase in the delay time. These findings are all consistent with the idea that NEM activates transport not by a direct action on the transport protein but by altering a phosphorylation-dephosphorylation cycle. The simplest hypothesis that is consistent with the data is that both cell swelling and NEM cause inhibition of a protein kinase. Kinase inhibition causes net dephosphorylation of some key substrate (not necessarily the transport protein); dephosphorylation of this substrate, probably by type 1 protein phosphatase, causes transport activation.

Residues of organochlorine pesticides, polychlorinated biphenyls (PCBs), and 16 elements were measured in American alligator (Alligator mississippiensis) eggs collected in 1984 from Lakes Apopka, Griffin, and Okeechobee in central and south Florida. Organochlorine pesticides were highest in eggs from Lake Apopka. None of the elements appeared to be present at harmful concentrations in eggs from any of the lakes. A larger sample of eggs was collected in 1985, but only from Lakes Griffin, a lake where eggs were relatively clean, and Apopka, where eggs were most contaminated. In 1985, hatching success of artificially incubated eggs was lower for Lake Apopka, and several organochlorine pesticides were higher than in eggs from Lake Griffin. However, within Lake Apopka, higher levels of pesticides in chemically analyzed eggs were not associated with reduced hatching success of the remaining eggs in the clutch. Therefore, it did not appear that any of the pesticides we measured were responsible for the reduced hatching success of Lake Apopka eggs.

No abstract

Transient extracellular pH changes accompany the exchange of chloride for sulfate across the erythrocyte membrane. The direction of the extracellular pH change during chloride efflux and sulfate influx depends on experimental conditions. When bicarbonate is present, the extracellular pH drops sharply at the outset of the anion exchange and tends to follow the partial ionic equilibrium described by Wilbrandt (W. Wilbrandt, 1942. Pfluegers Arch. 246:291). When bicarbonate is absent, however, the anion exchange causes the pH to rise, indicating that protons are cotransported with sulfate during chloride-sulfate exchange. The pH rise can be reversed by the addition of HCO(-3) (4 muM) or 2,4-dinitrophenol (90 muM). This demonstrates that the proton-sulfate cotransport can drive proton transport uphill. The stoichiometry of the transport is that one chloride exchanges for one sulfate plus one proton. These results support the titratable carrier model proposed by Gunn (Gunn, R.B. 1972, In: Oxygen Affinity of Hemoglobin and Red Cell Acid-Base Status. M. Rorth and P. Astrup, editors. p. 823. Munksgaard, Copenhagen) for erythrocyte membrane anion exchange.