Evidence for coupled transport of bicarbonate and sodium in cultured bovine corneal endothelial cells

Summary Usin gintracellular microelectrode technique, the response of the voltageV across the plasma membrane of cultured bovine corneal endothelial cells to changes in sodium and bicarbonate concentrations was investigated. (1) The electrical response to changes in [HCO ₃ ^– ] _o (depolarization upon lowering and hyperpolarization upon raising [HCO ₃ ^– ] _o ) was dependent on sodium. Lithium could fairly well be substituted for sodium, whereas potassium or choline were much less effective. (2) Removal of external sodium caused a depolarization, while a readdition led to a hyperpolarization, which increased with time of preincubation in the sodium-depleted medium. (3) The response to changes in [Na⁺] _o was dependent on bicarbonate. In a nominally bicarbonate-free medium, its amplitude was decreased or even reversed in sign. (4) Application of SITS or DIDS (10^–3 m) had a similar effect on the response to sodium as bicarbonate-depleted medium. (5) At [Na⁺] _o =151mm and [HCO ₃ ^– ] _o =46mm, the transients ofV depended, with 39.0±9.0 (sd) mV/decade, on bicarbonate and, with 15.3±5.8 (sd) mV/decade, on sodium. (6) After the preincubation of cells with lithium, replacement of Li by choline led to similar effects as the replacement of sodium by choline, though the response ofV was smaller with Li. This response could be reduced or reversed by the removal of bicarbonate or by the application of SITS. (7) Amiloride (10^–3 m) caused a reversible hyperpolarization of the steady-state potential by 8.5±2.6 mV (sd). It did not affect the immediate response to changes in [Na⁺] _o or [HCO ₃ ^– ] _o , but reduced the speed of regaining the steady-state potential after a change in [HCO ₃ ^– ] _o . (8) Ouabain (10^–4 m) caused a fast depolarization of –6.8±1.1 (sd) mV, which was followed by a continuing slower depolarization. The effect was almost identical at 10^–5 m. (9) It is suggested, that corneal endothelial cells possess a cotransport for sodium and bicarbonate, which transports net negative charage with these ions. It is inhibitable by stilbenes, but not directly affected by amiloride or ouabain. Lithium is a good substitute for sodium with respect to bicarbonate transport and is transported itself. In addition, the effect of amiloride provides indirect evidence for the existence of a Na⁺/H⁺-antiport. A model for the transepithelial transport of bicarbonate across the corneal endothelium is proposed.     

Ubiquitin-conjugating enzymes UBC4 and UBC5 mediate selective degradation of short-lived and abnormal proteins.

Ubiquitin-conjugating enzymes catalyse the covalent attachment of ubiquitin to target proteins. Members of this enzyme family are involved in strikingly diverse cellular functions: UBC2 (RAD6) is central to DNA repair, UBC3 (CDC34) is involved in cell cycle control. We have cloned the genes for two novel ubiquitin-conjugating enzymes, UBC4 and UBC5, from the yeast Saccharomyces cerevisiae. These enzymes mediate selective degradation of short-lived and abnormal proteins. UBC4 and UBC5 are closely related in sequence and complementing in function. Expression of UBC4 and UBC5 genes is heat inducible. UBC4 and UBC5 enzymes generate high mol. wt ubiquitin-protein conjugates in vivo consistent with previous studies which suggested that attachment of multiple ubiquitin molecules to proteolytic substrates is required for their selective degradation. UBC4 and UBC5 enzymes comprise a major part of total ubiquitin-conjugation activity in stressed cells. Turnover of short-lived proteins and canavanyl-peptides but not of long-lived proteins is markedly reduced in ubc4ubc5 mutants. Loss of UBC4 and UBC5 activity impairs cell growth, leads to inviability at elevated temperatures or in the presence of an amino acid analog, and induces the stress response.     

Noncanonical Role of the 9-1-1 Clamp in the Error-Free DNA Damage Tolerance Pathway

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Highlights? 9-1-1 and Exo1 are components of the error-free RAD6 pathway ? 9-1-1 promotes postreplicative template switching ? Polyubiquitylated PCNA and 9-1-1 cooperate in the error-free RAD6 pathway ? 9-1-1’s role in the error-free RAD6 pathway is uncoupled from checkpoint functions     

Common Gating of Both CLC Transporter Subunits Underlies Voltage-dependent Activation of the 2Cl?/1H+ Exchanger ClC-7/Ostm1

CLC anion transporters form dimers that function either as Cl⁻ channels or as electrogenic Cl⁻/H⁺ exchangers. CLC channels display two different types of “gates,” “protopore” gates that open and close the two pores of a CLC dimer independently of each other and common gates that act on both pores simultaneously. ClC-7/Ostm1 is a lysosomal 2Cl⁻/1H⁺ exchanger that is slowly activated by depolarization. This gating process is drastically accelerated by many CLCN7 mutations underlying human osteopetrosis. Making use of some of these mutants, we now investigate whether slow voltage activation of plasma membrane-targeted ClC-7/Ostm1 involves protopore or common gates. Voltage activation of wild-type ClC-7 subunits was accelerated by co-expressing an excess of ClC-7 subunits carrying an accelerating mutation together with a point mutation rendering these subunits transport-deficient. Conversely, voltage activation of a fast ClC-7 mutant could be slowed by co-expressing an excess of a transport-deficient mutant. These effects did not depend on whether the accelerating mutation localized to the transmembrane part or to cytoplasmic cystathionine-β-synthase (CBS) domains of ClC-7. Combining accelerating mutations in the same subunit did not speed up gating further. No currents were observed when ClC-7 was truncated after the last intramembrane helix. Currents and slow gating were restored when the C terminus was co-expressed by itself or fused to the C terminus of the β-subunit Ostm1. We conclude that common gating underlies the slow voltage activation of ClC-7. It depends on the CBS domain-containing C terminus that does not require covalent binding to the membrane domain of ClC-7.     

Effect of a soy protein diet on protein and energy metabolism and organ development in protein-restricted growing pigs

Two feeding experiments were carried out with castrated male pigs weighing between 10 and 30 kg to study acute and persisting dietary effects on growth and on protein and energy metabolism in growing pigs. Pigs were fed semi-synthetic isoenergetic and isonitrogenous diets at 50% protein requirement with either soy protein isolate (SPI) or casein (CAS) as sole protein source. Intake of protein and ME amounted to 9% w/w and 1800 kJ · kg BW ^{? 0.62} · d ^{? 1} in Exp. 1, respectively, and 9% w/w and 1430 kJ · kg BW ^{? 0.62} · d ^{? 1} in Exp. 2. The CAS diet was supplemented by Lys, Met, Thr and Trp. In Exp. 1 (acute effects), 18 pigs received the CAS diet for 24 days (period 1); 9 pigs were then switched to a SPI diet whereas 9 pigs continued on the CAS diet for another 31 days (period 2). In Exp. 2, a third period of 31 days was added in which the SPI group was switched back to CAS diet. The control group was fed on the CAS diet throughout Exp. 2 (86 days). Altogether the majority of parameters were not affected neither comparing SPI with CAS in Exp. 1 nor inspecting possible persistence of effects in Exp. 2. In detail, in Exp. 1 SPI compared to CAS feeding resulted in a lower efficiency of protein utilisation and lower protein retention. Attendant upon the lower protein retention an increased energy retention as fat was only observed in tendency. SPI feeding caused a decreased body weight, thyroid weight and increased hepatic carbohydrate content that persisted after the diet was changed back to CAS (Exp. 2).     

Patterns of island treeline elevation – a global perspective

Treeline research has strongly focused on mountain systems on the mainland. However, island treelines offer the opportunity to contribute to the global framework on treeline elevation due to their island‐specific attributes such as isolation, small area, low species richness and relative youth. We hypothesize that, similar to the mainland, latitude‐driven temperature variation is the most important determinant of island treeline elevation on a global scale. To test this hypothesis, we compared mainland with island treeline elevations. Then we focused 1) on the global effects of latitude, 2) on the regional effects of island type (continental vs oceanic islands) and 3) the local effects of several specific island characteristics (age, area, maximum island elevation, isolation and plant species richness). We collected a global dataset of islands (n = 86) by applying a stratified design using GoogleEarth and the Global Island Database. For each island we extracted data on latitude and local characteristics. Treeline elevation decreased from the mainland through continental to oceanic islands. Island treeline elevation followed a hump‐shaped latitudinal distribution, which is fundamentally different from the mainland double‐hump. Higher maximum island elevation generated higher treeline elevation and was found the best single predictor of island treeline elevation, even better than latitude. Lower island treeline elevation may be the result of a low mass elevation effect (MEE) influencing island climates and an increasingly impoverished species pool but also trade wind inversion‐associated aridity. The maximum island elevation effect possibly results from an increasing mass elevation effect (MEE) with increasing island elevation but also range shifts during climatic fluctuations and the summit syndrome (i.e. high wind speeds and poor soils in peak regions). Investigating islands in treeline research has enabled disentangling the global effect of latitude from regional and local effects and, at least for islands, a comprehensive quantification of the MEE.     

Methane production in cattle calculated by the nutrient composition of the diet

In this study data originating from complete metabolic trials with cattle of both sexes, fed 337 rations at feed intake levels between one to three times maintenance energy requirement were used to regress the total CH4 emission to the level of DM intake and to the nutrient composition, respectively. A major component of the measured CH4 emission cannot be explained by DM intake but is rather due to differences in dietary nutrient composition. The amount of digestible nutrients especially of the carbohydrate fraction (starch, sugar, N-free residuals) are reliable to estimate CH4 release with high precision (r2 = 0.885). Its production rate increased to 1.9-fold higher levels (range 1.8-2.1) per g of N-free residuals compared to that induced per g of protein, starch or sugar, respectively. Furthermore, diets rich in fat reduced CH4 formation in the rumen. The regression equations fit a wide range of diets and diet compositions, and more importantly, they are applicable to various types of production systems.