Modulation of water and urea transport in human red cells: Effects of pH and phloretin

Summary It has previously been shown by Macey and Farmer (Biochim. Biophys. Acta 211:104–106, 1970) that phloretin inhibits urea transport across the human red cell membrane yet has no effect on water transport. Jennings and Solomon (J. Gen. Physiol. 67:381–397, 1976) have shown that there are separate lipid and protein binding sites for phloretin on the red cell membrane. We have now found that urea transport is inhibited by phloretin binding to the lipids with aK ₁ of 25±8 m in reason-able agreement with theK _D of 54±5 m for lipid binding. These experiments show that lipid/protein interactions can alter the conformational state of the urea transport protein. Phloretin binding to the protein site also modulates red cell urea transport, but the modulation is opposed by the specific stilbene anion transport inhibitor, DIDS (4,4-diisothiocyano-2,2-stilbene disulfonate), suggesting a linkage between the urea transport protein and band 3. Neither the lipid nor the protein phloretin binding site has any significant effect on water transport. Water transport is, however, inhibited by up to 30% in a pH-dependent manner by DIDS binding, which suggests that the DIDS/band 3 complex can modulate water transport.     

Osmotic properties of human red cells

Summary When an osmotic pressure gradient is applied to human red cells, the volume changes anomalously, as if there were a significant fraction of nonosmotic water which could not serve as solvent for the cell solutes, a finding which has been discussed widely in the literature. In 1968, Gary-Bobo and Solomon (J. Gen. Physiol. 52:825) concluded that the anomalies could not be entirely explained by the colligative properties of hemoglobin (Hb) and proposed that there was an additional concentration dependence of the Hb charge (z_Hb). A number of investigators, particularly Freedman and Hoffman (1979,J. Gen. Physiol. 74:157) have been unable to confirm Gary-Bobo and Solomon's experimental evidence for this concentration dependence of z_Hb and we now report that we are also unable to repeat the earlier experiments. Nonetheless, there still remains a significant anomaly which amounts to 12.5±0.8% of the total isosmotic cell water (P0.0005,t test), even after taking account of the concentration dependence of the Hb osmotic coefficient and all the other known physical chemical constraints, ideal and nonideal. It is suggested that the anomalies at high Hb concentration in shrunken cells may arise from the ionic strength dependence of the Hb osmotic coefficient. In swollen red cells at low ionic strength, solute binding to membrane and intracellular proteins is increased and it is suggested that this factor may account, in part, for the anomalous behavior of these cells.     

The colliculus superior modulates ACTH-induced excessive grooming

In previous studies the involvement of nigrostriatal dopaminergic activity in ACTH(1-24)-induced grooming has been established. It was suggested that the dopaminergic modulation of ACTH(1-24)-induced excessive grooming is exerted through the striato-nigro-collicular pathway. To obtain further evidence it was investigated, whether local application of GABAergic agents into the colliculus superior modulates excessive grooming occurring after an intraventricular injection with ACTH(1-24). It appeared that intra-collicular picrotoxin (a GABAergic antagonist) suppressed ACTH-induced grooming, whereas muscimol (a GABAergic agonist) enhanced the grooming response. The picrotoxin-induced R(unning) F(it) B(ehavior), elicited from the colliculus superior was also seen after intraventricular administration of picrotoxin. A detailed comparison of this behavioral response seen after both routes of administration of picrotoxin suggests that intraventricularly injected picrotoxin may well induce the RFB via a direct effect on the colliculus superior. Lesions placed in the colliculus superior completely abolished picrotoxin-induced RFB, exploration and orientation behavior. Yet, these lesions did not reduce excessive grooming suggesting that although this region may be involved in the modulation of ACTH-induced grooming it is not the primary site of peptide action.     

The influence of Ca2+ on the turbidity of DPPC-DMPA vesicles within the temperature range of the phase transition

The influence of the addition of Ca2+ on the phase behaviour of vesicles, composed of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidic acid (DMPA) in a ratio of 4 to 1, has been investigated by means of turbidity measurements. As expected one single phase transition for the mixed phospholipids was observed in the absence of Ca2+. Passing through the temperature range of this transition after the addition of Ca2+, conditions appeared to favor fusion of the vesicles. A possible reason for this is that during the transition Ca2+ may permeate through the vesicle membranes and gain access to the inside DMPA binding sites. Therefore it is not unambiguously possible to determine phase transition temperatures from the turbidity changes that occur under these conditions. However, when within the temperature range of the phase transition of the mixed phospholipids the influence of Ca2+ addition to the vesicles was recorded isothermally, at each temperature separately, the final plot of turbidity versus temperature turned out to be far less confused by fusion events and adopted the form of two separate phase transitions. The temperatures at which these two transitions occur closely resemble the phase transition temperatures that may be observed in the absence of Ca2+ for DMPA and DPPC alone, 39 degrees C and 43 degrees C respectively. The results of this study suggest that when Ca2+ has only access to the outside of the vesicle membranes it may segregate the neutral and the acidic phospholipids into separate domains, both domains adopting their proper phase condition at the actual temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

A combined CaMKII inhibition and mineralocorticoid receptor antagonism via eplerenone inhibits functional deterioration in chronic pressure overloaded mice

In the diseased and remodelled heart, increased activity and expression of Ca^2+/calmodulin‐dependent protein kinase II (CaMKII), an excess of fibrosis, and a decreased electrical coupling and cellular excitability leads to disturbed calcium homeostasis and tissue integrity. This subsequently leads to increased arrhythmia vulnerability and contractile dysfunction. Here, we investigated the combination of CaMKII inhibition (using genetically modified mice expressing the autocamtide‐3‐related‐peptide (AC3I)) together with eplerenone treatment (AC3I‐Epler) to prevent electrophysiological remodelling, fibrosis and subsequent functional deterioration in a mouse model of chronic pressure overload. We compared AC3I‐Epler mice with mice only subjected to mineralocorticoid receptor (MR) antagonism (WT‐Epler) and mice with only CaMKII inhibition (AC3I‐No). Our data show that a combined CaMKII inhibition together with MR antagonism mitigates contractile deterioration as was manifested by a preservation of ejection fraction, fractional shortening, global longitudinal strain, peak strain and contractile synchronicity. Furthermore, patchy fibrosis formation was reduced, potentially via inhibition of pro‐fibrotic TGF‐β/SMAD3 signalling, which related to a better global contractile performance and a slightly depressed incidence of arrhythmias. Furthermore, the level of patchy fibrosis appeared significantly correlated to eplerenone dose. The addition of eplerenone to CaMKII inhibition potentiates the effects of CaMKII inhibition on pro‐fibrotic pathways. As a result of the applied strategy, limiting patchy fibrosis adheres to a higher synchronicity of contraction and an overall better contractile performance which fits with a tempered arrhythmogenesis.     

Role of Adenosine Kinase in the Biological (Antiviral and Anticellular) Activities of Adenosine Analogues

Abstract

A paired adenosine kinase-positive/adenosine kinase-negative cell system is proposed to distinguish those adenosine analogues that need to be phosphorylated to exert their biological effects from those that are mainly targeted at S-adenosyl-L-homocysteine hydrolase.     

Structure of a bacterial type IV secretion core complex at subnanometre resolution

Type IV secretion (T4S) systems are able to transport DNAs and/or proteins through the membranes of bacteria. They form large multiprotein complexes consisting of 12 proteins termed VirB1‐11 and VirD4. VirB7, 9 and 10 assemble into a 1.07 MegaDalton membrane‐spanning core complex (CC), around which all other components assemble. This complex is made of two parts, the O‐layer inserted in the outer membrane and the I‐layer inserted in the inner membrane. While the structure of the O‐layer has been solved by X‐ray crystallography, there is no detailed structural information on the I‐layer. Using high‐resolution cryo‐electron microscopy and molecular modelling combined with biochemical approaches, we determined the I‐layer structure and located its various components in the electron density. Our results provide new structural insights on the CC, from which the essential features of T4S system mechanisms can be derived.     

A new family of small, palmitoylated, membrane-associated proteins, characterized by the presence of a cysteine-rich hydrophobic motif

We recently cloned the CHIC2 gene (previously BTL) by virtue of its involvement in a chromosomal translocation t(4;12)(q11;p13) occurring in acute myeloid leukemias. In this study we show that CHIC2 is a member of a highly conserved family of proteins characterized by the presence of a striking cysteine-rich hydrophobic (CHIC) motif. Our data illustrate that cysteines in this central CHIC motif are palmitoylated and that CHIC2 is associated with vesicular structures and the plasma membrane. The CHIC proteins thus resemble the cysteine string proteins, which function in regulated exocytosis.