Transport of chlorine in the proximal tubule. Its effects on water-electrolyte absorption

Several studies in rat kidney have established that an appreciable fraction of proximal absorption is passive in nature and occurs across the highly conductive paracellular pathway. Passive absorption is generally ascribed to the transepithelial Cl- distribution, luminal Cl- activity (alpha lCl) being higher than plasma Cl- activity (alpha pCl). The inequality alpha lCl greater than alpha pCl generates a transepithelial diffusion potential, lumen positive, which taken together with the chemical potential differences of Cl- and Na+ across the epithelium gives rise to transepithelial electrochemical potential differences for Cl- and Na+ favoring their absorption. The alpha lCl greater than alpha pCl distribution is traditionally ascribed to preferential bicarbonate absorption. We argue that HCO3- absorption alone cannot generate a non equilibrium transepithelial Cl- distribution. Other mechanisms are necessary. Our measurements in amphibian proximal tubule demonstrate that the intracellular Cl- activity, alpha cCl, is higher than the theoretical value predicted for equilibrium. This distribution is the result of two basolateral coupled transport processes (Cl-/HCO3- exchange and Cl-/Na+ cotransport). It contributes to the exit of Cl- from cell to lumen (by passive diffusion and K+/Cl- cotransport), yielding alpha lCl values higher than the theoretical value for equilibrium with regard to plasma. Thus, a small transcellular flux of Cl- (without solvent) proceeds from interstitium to lumen. It compensates the dissipative tendency of a much higher paracellular Cl- absorptive flux (in association with water) on the transepithelial Cl- gradient. The result is a steady-state luminal Cl- distribution above equilibrium, along the major part of the proximal tubule.     

Transformation and Transduction in Recombination-defective Mutants of Bacillus subtilis

The effects on transformation and transduction of an ultraviolet sensitivity (uvr(-)) and two ultraviolet sensitivity-recombination deficiency (rec-1(-) and rec-2(-)) mutations in isogenic strains of Bacillus subtilis were investigated. Transformation frequency in the rec-1(-) and rec-2(-) strains was reduced to approximately 5 and 25%, respectively, of the parental strains. Normal kinetics of deoxyribonucleic acid dose response in transformation were found for the rec-1(+) and rec-2(-) strains. Biphasic curves were obtained with the rec-1(-) strains. Transduction frequency with bacteriophage SP-10 decreased parallel to transformation frequency in the rec-1(-) and rec-2(-) strains. This result suggests that transformation and SP-10 transduction share a common mechanism for genetic recombination. It also indicates that the reduction in transformation frequency of these strains was not due to altered competence. Transduction frequency with bacteriophage PBS-1 or 3NT, on the contrary, was not diminished in rec-1(-) strains. This frequency was reduced in rec-2(-) strains but not as severely as that of transformation or SP-10 transduction. Several hypotheses to interpret these differences are presented. Recombination frequency between linked markers was reduced more than 50% in transformation by the presence of the rec-1(-) mutation. Linkage was unaffected in the rec-2(-) strains. Neither the rec-1(-) nor the rec-2(-) mutation had an effect on linkage in PBS-1 or 3NT transduction. The uvr(-) strains were transformed at a frequency equal to or greater than that of the parental strains. These strains were transduced by all bacteriophage systems at frequencies about twofold higher than those of parental strains.     

Shoot Sr concentrations in relation to shoot Ca concentrations and to soil properties

This work was aimed to investigate whether shoot Sr concentrations of plant species are related to respective Ca concentrations and to soil properties and to compare the Sr-Ca observed ratios (OR), defined as the quotient of the ratios Sr/Ca in shoots and in the soil solution or in the extractable form, among species and soils. Ten pasture plant species were grown in pots (1-L volume) filled with eight soils differing in the various physicochemical characteristics. Each pot received 50 mg Sr except those of the soil with the highest cation exchange capacity (C.E.C.) that received 100 mg Sr per pot. For each soil, shoot Sr concentrations of species were linearly and positively related with the respective Ca concentrations. C.E.C, organic matter content and Ca in the soil solution or in the extractable form were the only soil properties that were related, all negatively, with shoot Sr concentrations. The ratio of extractable Sr and Ca was positively and linearly related with the ratio of Sr and Ca. in the soil solution. OR was affected by both species and soils. Most of OR values of all species in all soils ranged between 0.8 and 1.5, except for the grass Agrostis capillaris which had the highest values for most of soils. This indicates that Agrostis capillaris compared to other species, takes up proportionally more Sr than Ca.     

Genetic Structure and Internal Rearrangements of Stable Merodiploids from BACILLUS SUBTILIS Strains Carrying the trpE26 Mutation

Transformation and transduction to tryptophan independence of strains of Bacillus subtilis carrying the "trpE26" chromosomal aberrations (a translocation and an inversion) with a "normal" 168 type strain as donor induce a tandem duplication of the thrA-ilvA region of the chromosome. The clones possessing this unstable duplication segregate besides the Trp^- some stable Trp⁺ cells which retain only part of the duplication (the trpE-ilvA region) in nontandem configuration. Such clones may also be produced directly during the crosses. The genetic map of these clones (designated as class I stable merodiploids) was constructed: they possess the tranlocation and the inversion of the trpE26 parental strain. Another type of stable Trp⁺ clones (class II) also appears, although more rarely, in similar crosses. Studies on their genetic structure revealed that they are haploid for the trpE-ilvA region and carry a nontandem duplication of the thrA-trpE region. In these clones the cysB-tre region has the orientation of the 168 type strain. The duplications in both classes are stable, that of class I being more stable than that of class II where loss of one copy of the thrA-trpE region leads to about 1% haploid cells. Detailed genetic studies on heterozygous clones from both classes have shown exchange of alleles between copies of the nontandem duplications. Models are proposed for the formation of each class of merodiploids and for recombination events taking place in them. These models imply recombination at sequences of intrachromosomal homology and (or) introduction of heterologous juncions ("novel joints") by transformation or transduction.     

Chemostat adaptation of Escherichia coli B/r/1 to low water activity

Chemostat cultures of Escherichia coli B/r/1 under conditions of glucose limitation in a salts medium at high water activity ( a _w of 0.999) and at an a _w of 0.987, controlled by NaCl, have been compared. The system was run at dilution rates above 0.035 h^-1. The results suggested that the organism adapted to the lower a _w of the medium as no significant change was observed in the energy requirement for maintenance, although the maximum molar growth yield for glucose decreased by 23.2%. Such cultures showed also a shorter lag and a higher growth rate in batch at an a _w of 0.987, as compared with cultures initiated with an unadapted ( a _w of 0.999) inoculum.     

Chloride distribution in the proximal convoluted tubule ofNecturus kidney

Summary To assess the mechanism(s) by which intraluminal chloride concentration is raised above equilibrium values, intracellular Cl^– activity ( _i ^Cl ) was studied in the proximal tubule ofNecturus kidney. Paired measurements of cell membrane PD (V _BL) and Cl-selective electrode PD (V _BL ^Cl ) were performed in single tubules, during reversible shifts of peritubular or luminal fluid composition. Steadystate _i ^Cl was estimated at 14.6±0.6 mmol/liter, a figure substantially higher than that predicted for passive distribution. To determine the site of the uphill Cl^– transport into the cell, an inhibitor of anion transport (SITS) was added to the perfusion fluid. Introduction of SITS in peritubular perfusate decreased _i ^Cl , whereas addition of the drug in luminal fluid slightly increased _i ^Cl ; both results are consistent with basolateral membrane uphill Cl^– transport from interstitium to the cell. TMA⁺ for Na⁺ substitutions in either luminal or peritubular perfusate had no effect on _i ^Cl . Removal of bicarbonate from peritubular fluid, at constant pH (a situation increasing HCO ₃ ^– outflux), resulted in an increase of _i ^Cl , presumably related to enhanced Cl^– cell influx: we infer that Cl^– is exchanged against HCO ₃ ^– at the basolateral membrane. The following mechanism is suggested to account for the rise in luminal Cl^– concentration above equilibrium values: intracellular CO₂ hydration gives rise to cell HCO ₃ ^– concentrations above equilibrium. The passive exit of HCO ₃ ^– at the basolateral membrane energizes an uphill entry of Cl^– into the cell. The resulting increase of _i ^Cl , above equilibrium, generates downhill Cl^– diffusion from cell to lumen. As a result, luminal Cl^– concentration also increases.C.N.R.S. Greco 24. Part of this work was presented at the 12^th annual meeting of the American Society of Nephrology, Boston, Mass. (Edelman et al., 1979).     

Substrate specificity and adenosine triphosphatase activity of the ATP-dependent deoxyribonuclease of Bacillus subtilis

Studies on the specificity of the ATP-dependent DNase of Bacillus subtilis 168, carried out with pure enzyme at the optimal conditions for its action, have shown that the substrate is double-stranded linear DNA. Linear single-stranded DNA (separated strands of B. subtilis DNA and linear phage fd DNA) is not attacked, neither are there any circular forms (supercoiled or nicked simian virus 40 and circular single-stranded fd DNAs). The double-stranded DNA can be completely hydrolysed, the limit products being, almost exclusively, mononucleotides. The presence of terminal phosphate residues in the substrate (either at the 3' or the 5' end) is not necessary for enzyme action. This DNase appears therefore to be an exonuclease processively liberating mononucleotides from both strands of the native linear DNA. ATP (indispensable for the DNase reaction) is also hydrolysed by the enzyme, to ADP and inorganic orthophosphate (Pi) in the presence of DNA. The apparent Km for ATP, in the ATPase reaction, is 0.15 mM. At high ATP concentrations, which inhibit the DNase activity, there is activation of the ATPase reaction. Three molecules of ATP are consumed for each DNA phosphodiester bond split, at optimal conditions for DNase activity.     

Growth of Escherichia coli B/r/l in a semi-continuous system designed for the synchronization of cell division

Escherichia coli B/r/l was grown under conditions of periodic feeding. Glucose, the only carbon source, was supplied at intervals longer than the generation time of the organism. Thus, each period of glucose availability was followed by a period of depletion. This process gave rise to two synchronous populations, one dividing shortly after a new supply of fresh medium and the other dividing at a later stage within the feeding cycle. Thymine incorporation experiments suggested that the double population emerged as the result of a discriminatory blockage of DNA replication.     

Identification of membrane transport processes in renal cells, by means of liquid ion exchanger microelectrodes

The development of liquid-ion-exchanger microelectrodes displaying tip diameters less than or equal to 1 micron has permitted direct measurement of the transmembrane chemical and electrochemical gradients of several permeant ion species in cells of small size. We have used Cl- and H+ resins to study the intracellular Cl- activity (alpha iCl) and cell pH (pHi) in the proximal tubule of Necturus kidney. These determinations were performed in association with perfusion of peritubular capillaries by several artificial solutions, in order to assess the dependence of alpha iCl and pHi on the composition of physiologic plasma constituents and selected inhibitors. The main findings are: Intracellular chloride activity, alpha iCl, is higher than the theoretical value predicted from electrochemical equilibrium. Peritubular application of SITS resulted in a decrease of alpha iCl and increase of pHi; these observations are taken to indicate that Cl- uptake is achieved across the basolateral membrane in exchange for HCO-3 by a mechanism sensitive to SITS. Na+ removal from peritubular fluid elicited a small reduction of alpha iCl, suggesting the presence of carrier-mediated Cl--Na+ cotransport from interstitium to cell, contributing to the rise of alpha iCl above equilibrium. In conclusion, two carrier-mediated processes (Cl-/HCO-3 exchange and Cl--Na+ symport) located at the basolateral membrane of the proximal tubule may account for the establishment of alpha iCl values above equilibrium, at steady state. The physiologic role of these carriers is discussed in relation to proximal electrolyte absorption.     

Differential microRNA Profiles and Their Functional Implications in Different Immunogenetic Subsets of Chronic Lymphocytic Leukemia

Critical processes of B-cell physiology, including immune signaling through the B-cell receptor (BcR) and/or Toll-like receptors (TLRs), are targeted by microRNAs. With this in mind and also given the important role of BcR and TLR signaling and microRNAs in chronic lymphocytic leukemia (CLL), we investigated whether microRNAs could be implicated in shaping the behavior of CLL clones with distinct BcR and TLR molecular and functional profiles. To this end, we examined 79 CLL cases for the expression of 33 microRNAs, selected on the following criteria: (a) deregulated in CLL versus normal B-cells; (b) differentially expressed in CLL subgroups with distinct clinicobiological features; and, (c) if meeting (a) + (b), having predicted targets in the immune signaling pathways. Significant upregulation of miR-150, miR-29c, miR-143 and miR-223 and downregulation of miR-15a was found in mutated versus unmutated CLL, with miR-15a showing the highest fold difference. Comparison of two major subsets with distinct stereotyped BcRs and signaling signatures, namely subset 1 [IGHV1/5/7-IGKV1(D)-39, unmutated, bad prognosis] versus subset 4 [IGHV4-34/IGKV2-30, mutated, good prognosis] revealed differences in the expression of miR-150, miR-29b, miR-29c and miR-101, all down-regulated in subset 1. We were also able to link these distinct microRNA profiles with cellular phenotypes, importantly showing that, in subset 1, miR-101 downregulation is associated with overexpression of the enhancer of zeste homolog 2 (EZH2) protein, which has been associated with clinical aggressiveness in other B-cell lymphomas. In conclusion, specific miRNAs differentially expressed among CLL subgroups with distinct BcR and/or TLR signaling may modulate the biological and clinical behavior of the CLL clones.