Hemoglobin Fontainebleau [a21(B2)Ala>Pro]: The second report from India

Structural hemoglobin (Hb) variants are mainly due to point mutations in the globin genes resulting in single amino acid substitutions. Until date, about 200 alpha chain variants have been identified and they are usually detected during the hemoglobinopathy screening programs. Under a community control program for hemoglobinopathies, which involved screening of antenatal cases followed by prenatal diagnosis if indicated. Here, we report a rare alpha globin gene variant Hb Fontainebleau [a21(B2)Ala>Pro] detected in the heterozygous condition in a 35-year-old pregnant lady screened during this program. This is the second report of this alpha globin variant from India. Unlike the earlier case from India where Hb Fontainebleau was reported in a neonate who was also a carrier of Hb Sickle and had no clinical problems, this case presented with a bad obstetric history associated with the secondary infertility. However, the presence of the variant and the obstetric complications may be unrelated.     

Bcl-2 acts subsequent to and independent of Ca fluxes to inhibit apoptosis in thapsigargin- and glucocorticoidmtreated mouse lymphoma cells

The mechanism by which Bcl-2 inhibits apoptosis is unknown. One proposal is that Bcl-2 regulates intracellular Ca²⁺ fluxes thought to mediate apoptosis. In the present study, we investigated Bcl-2's mechanism of action by determining the effect of Bcl-2 on intracellular Ca²⁺ fluxes in the WEH17.2 mouse lymphoma cell line, which does not express Bcl-2, and its stable transfectant, which expresses a high level of Bcl-2. Treatment with the endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin produced marked alterations in intracellular Ca²⁺ homeostasis in both WEH17.2 and W.Hb12 cells, including elevation of free cytosolic Ca²⁺, endoplasmic reticulum Ca²⁺ pool depletion, capacitative entry of extracellular Ca²⁺, and increased loading of Ca²⁺ into mitochondria. Similar changes in intracellular Ca²⁺ occurred spontaneously in both cell lines following exponential growth. In both situations, W.Hb12 cells maintained optimal viability despite marked alterations in intracellular Ca ²⁺' whereas WEH17.2 cells underwent apoptosis. Treatment with the glucocorticoid hormone, dexamethasone, induced apoptosis in WEH17.2 cells, but not in W.HB12 cells, even though dexamethasone treatment did not alter intracellular Ca²⁺ homeostasis in either cell line. These findings indicate that Bcl-2 acts downstream from intracellular Ca ²⁺ fluxes in a pathway where Ca²⁺-dependent and Ca²⁺-independent death signals converge.     

The GluCre-ROSA26EYFP mouse: a new model for easy identification of living pancreatic alpha-cells

The control of glucagon secretion by pancreatic alpha-cells is poorly understood, largely because of the difficulty to recognize living alpha-cells. We describe a new mouse model, referred to as GluCre-ROSA26EYFP (or GYY), allowing easy alpha-cell identification because of specific expression of EYFP. GYY mice displayed normal glycemic control during a fasting/refeeding test or intraperitoneal insulin injection. Glucagon secretion by isolated islets was normally inhibited by glucose and stimulated by adrenaline. [Ca(2+)](c) responses to arginine, adrenaline, diazoxide and tolbutamide, were similar in GYY and control mice. Hence, this new mouse model is a reliable and powerful tool to specifically study alpha-cells.     

The role of organ of Corti mass in passive cochlear tuning

The mechanism for passive cochlear tuning remains unsettled. Early models considered the organ of Corti complex (OCC) as a succession of spring-mass resonators. Later, traveling wave models showed that passive tuning could arise through the interaction of cochlear fluid mass and OCC stiffness without local resonators. However, including enough OCC mass to produce local resonance enhanced the tuning by slowing and thereby growing the traveling wave as it approached its resonant segment. To decide whether the OCC mass plays a role in tuning, the frequency variation of the wavenumber of the cochlear traveling wave was measured (in vivo, passive cochleae) and compared to theoretical predictions. The experimental wavenumber was found by taking the phase difference of basilar membrane motion between two longitudinally spaced locations and dividing by the distance between them. The theoretical wavenumber was a solution of the dispersion relation of a three-dimensional cochlear model with OCC mass and stiffness as the free parameters. The experimental data were only well fit by a model that included OCC mass. However, as the measurement position moved from a best-frequency place of 40 to 12 kHz, the role of mass was diminished. The notion of local resonance seems to only apply in the very high-frequency region of the cochlea.     

Tangential flow filtration of hemoglobin

Bovine and human hemoglobin (bHb and hHb, respectively) was purified from bovine and human red blood cells via tangential flow filtration (TFF) in four successive stages. TFF is a fast and simple method to purify Hb from RBCs using filtration through hollow fiber (HF) membranes. Most of the Hb was retained in stage III (100 kDa HF membrane) and displayed methemoglobin levels less than 1%, yielding final concentrations of 318 and 300 mg/mL for bHb and hHb, respectively. Purified Hb exhibited much lower endotoxin levels than their respective RBCs. The purity of Hb was initially assessed via SDS‐PAGE, and showed tiny impurity bands for the stage III retentate. The oxygen affinity (P₅₀) and cooperativity coefficient (n) were regressed from the measured oxygen‐RBC/Hb equilibrium curves of RBCs and purified Hb. These results suggest that TFF yielded oxygen affinities of bHb and hHb that are comparable to values in the literature. LC‐MS was used to measure the molecular weight of the alpha (α) and beta (β) globin chains of purified Hb. No impurity peaks were present in the HPLC chromatograms of purified Hb. The mass of the molecular ions corresponding to the α and β globin chains agreed well with the calculated theoretical mass of the α‐ and β‐ globin chains. Taken together, our results demonstrate that HPLC‐grade Hb can be generated via TFF. In general, this method can be more broadly applied to purify Hb from any source of RBCs. This work is significant, since it outlines a simple method for generating Hb for synthesis and/or formulation of Hb‐based oxygen carriers. © 2008 American Institute of Chemical Engineers, 2009     

Alterations in striatal synaptic transmission are consistent across genetic mouse models of Huntington's disease

Since the identification of the gene responsible for HD (Huntington''s disease), many genetic mouse models have been generated. Each employs a unique approach for delivery of the mutated gene and has a different CAG repeat length and background strain. The resultant diversity in the genetic context and phenotypes of these models has led to extensive debate regarding the relevance of each model to the human disorder. Here, we compare and contrast the striatal synaptic phenotypes of two models of HD, namely the YAC128 mouse, which carries the full-length huntingtin gene on a yeast artificial chromosome, and the CAG140 KI (knock-in) mouse, which carries a human/mouse chimaeric gene that is expressed in the context of the mouse genome, with our previously published data obtained from the R6/2 mouse, which is transgenic for exon 1 mutant huntingtin. We show that striatal MSNs (medium-sized spiny neurons) in YAC128 and CAG140 KI mice have similar electrophysiological phenotypes to that of the R6/2 mouse. These include a progressive increase in membrane input resistance, a reduction in membrane capacitance, a lower frequency of spontaneous excitatory postsynaptic currents and a greater frequency of spontaneous inhibitory postsynaptic currents in a subpopulation of striatal neurons. Thus, despite differences in the context of the inserted gene between these three models of HD, the primary electrophysiological changes observed in striatal MSNs are consistent. The outcomes suggest that the changes are due to the expression of mutant huntingtin and such alterations can be extended to the human condition.     

Characterization of conformational and dynamic properties of natively unfolded human and mouse alpha-synuclein ensembles by NMR: implication for aggregation

Conversion of human α-synuclein (aS) from the free soluble state to the insoluble fibrillar state has been implicated in the etiology of Parkinson's disease. Human aS is highly homologous in amino acid sequence to mouse aS, which contains seven substitutions including the A53T that has been linked to familial Parkinson's disease, and including five substitutions in the C-terminal region. It has been shown that the rate of fibrillation is highly dependent on the exact sequence of the protein, and mouse aS is reported to aggregate more rapidly than human aS in vitro. Nuclear magnetic resonance experiments of mouse and human aS at supercooled temperatures (263 K) are used to understand the effect of sequence on conformational fluctuations in the disordered ensembles and to relate these to differences in propensities to aggregate. We show that both aS are natively unfolded at low temperature with different propensities to secondary structure, backbone dynamics and long-range contacts across the protein. Mouse aS exhibits a higher propensity to helical conformation around the C-terminal substitutions as well as the loss of transient long-range contacts from the C- to the N-terminal end and hydrophobic central regions of the protein relative to human aS. Lack of back-folding from the C-terminal end of mouse aS exposes the N-terminal region, which is shown, by ¹⁵N relaxation experiments, to be very restricted in mobility relative to human aS. We propose that the restricted mobility in the N-terminal region may arise from transient interchain interactions, suggesting that the N-terminal KTK(E/Q)GV repeats may serve as initiation sites for aggregation in mouse aS. These transient interchain interactions coupled with a non-Aβ amyloid component (NAC) region that is both more exposed and has a higher propensity to β structure may accelerate the rate of fibril formation of aS.     

Activation of PI3Kα by physiological effectors and by oncogenic mutations: structural and dynamic effects

PI3Kα, a heterodimeric lipid kinase, catalyzes the conversion of phosphoinositide-4,5-bisphosphate (PIP₂) to phosphoinositide-3,4,5-trisphosphate (PIP₃), a lipid that recruits to the plasma membrane proteins that regulate signaling cascades that control key cellular processes such as cell proliferation, carbohydrate metabolism, cell motility, and apoptosis. PI3Kα is composed of two subunits, p110α and p85, that are activated by binding to phosphorylated receptor tyrosine kinases (RTKs) or their substrates. The gene coding for p110α, PIK3CA, has been found to be mutated in a large number of tumors; these mutations result in increased PI3Kα kinase activity. The structure of the complex of p110α with a fragment of p85 containing the nSH2 and the iSH2 domains has provided valuable information about the mechanisms underlying the physiological activation of PI3Kα and its pathological activation by oncogenic mutations. This review discusses information derived from x-ray diffraction and theoretical calculations regarding the structural and dynamic effects of mutations in four highly mutated regions of PI3K p110α, as well as the proposed mechanisms by which these mutations increase kinase activity. During the physiological activation of PI3Kα, the phosphorylated tyrosine of RTKs binds to the nSH2 domain of p85, dislodging an inhibitory interaction between the p85 nSH2 and a loop of the helical domain of p110α. Several of the oncogenic mutations in p110α activate the enzyme by weakening this autoinhibitory interaction. These effects involve structural changes as well as changes in the dynamics of the enzyme. One of the most common p110α mutations, H1047R, activates PI3Kα by a different mechanism: it increases the interaction of the enzyme with the membrane, maximizing the access of the PI3Kα to its substrate PIP₂, a membrane lipid.     

Structural and functional determinants of conserved lipid interaction domains of inward rectifying Kir6.2 channels

All members of the inward rectifiier K(+) (Kir) channel family are activated by phosphoinositides and other amphiphilic lipids. To further elucidate the mechanistic basis, we examined the membrane association of Kir6.2 fragments of K(ATP) channels, and the effects of site-directed mutations of these fragments and full-length Kir6.2 on membrane association and K(ATP) channel activity, respectively. GFP-tagged Kir6.2 COOH terminus and GFP-tagged pleckstrin homology domain from phospholipase C delta1 both associate with isolated membranes, and association of each is specifically reduced by muscarinic m1 receptor-mediated phospholipid depletion. Kir COOH termini are predicted to contain multiple beta-strands and a conserved alpha-helix (residues approximately 306-311 in Kir6.2). Systematic mutagenesis of D307-F315 reveals a critical role of E308, I309, W311 and F315, consistent with residues lying on one side of a alpha-helix. Together with systematic mutation of conserved charges, the results define critical determinants of a conserved domain that underlies phospholipid interaction in Kir channels.     

The effect of fructan on membrane lipid organization and dynamics in the dry state

Fructans are a group of fructose-based oligo- and polysaccharides, which appear to be involved in membrane preservation during dehydration by interacting with the membrane lipids. To get further understanding of the protective mechanism, the consequences of the fructan-membrane lipid interaction for the molecular organization and dynamics in the dry state were studied. POPC and DMPC were investigated in the dry state by (2)H, (31)P NMR, and Fourier transform infrared spectroscopy using two types of fructan and dextran. The order-disorder transition temperature of dry POPC was reduced by 70 degrees C in the presence of fructan. Fructan increased the mobility of the acyl chains, but immobilized the lipid headgroup region. Most likely, fructans insert between the headgroups of lipids, thereby spacing the acyl chains. This results in a much lower phase transition temperature. The headgroup is immobilized by the interaction with fructan. The location of the interaction with the lipid headgroup is different for the inulin-type fructan compared to the levan-type fructan, since inulin shows interaction with the lipid phosphate group, whereas levan does not. Dextran did not influence the phase transition temperature of dry POPC showing that reduction of this temperature is not a general property of polysaccharides.     

Regulation of cytosolic and mitochondrial ATP levels in mouse eggs and zygotes

Fertilization activates development by stimulating a plethora of ATP consuming processes that must be provided for by an up-regulation of energy production in the zygote. Sperm-triggered Ca²⁺ oscillations are known to be responsible for the stimulation of both ATP consumption and ATP supply but the mechanism of up regulation of energy production at fertilization is still unclear. By measuring [Ca²⁺] and [ATP] in the mitochondria of fertilized mouse eggs we demonstrate that sperm entry triggers Ca²⁺ oscillations in the cytosol that are transduced into mitochondrial Ca²⁺ oscillations pacing mitochondrial ATP production. This results, during fertilization, in an increase in both [ATP]_mito and [ATP]_cyto. We also observe the stimulation of ATP consumption accompanying fertilization by monitoring [Ca²⁺]_cyto and [ATP]_cyto during fertilization of starved eggs. Our observations reveal that lactate, in contrast to pyruvate, does not fuel mitochondrial ATP production in the zygote. Therefore lactate-derived pyruvate is somehow diverted from mitochondrial oxidation and may be channeled to other metabolic routes. Together with our earlier findings, this study confirms the essential role for exogenous pyruvate in the up-regulation of ATP production at the onset of development, and suggests that lactate, which does not fuel energetic metabolism may instead regulate the intracellular redox potential.     

Extracellular Ca²+ alleviates NaCl-induced stomatal opening through a pathway involving H₂O₂-blocked Na+ influx in Vicia guard cells

To gain further insights into the function of extracellular Ca2+ in alleviating salt stress, Vicia faba guard cell protoplasts (GCPs) were patch-clamped in a whole-cell configuration. The results showed that 100 mM NaCl clearly induced Na+ influx across the plasma membrane in GCPs and promoted stomatal opening. Extracellular Ca2+ at 10 mM efficiently blocked Na+ influx and inhibited stomatal opening, which was partially abolished by La3+ (an inhibitor of plasma membrane Ca2+ channel) or catalase (CAT, a H?O? scavenger), respectively. These results suggest that the plasma membrane Ca2+ channels and H?O? possibly mediate extracellular Ca2+-blocked Na+ influx in GCPs. Furthermore, extracellular Ca2+ activated the plasma membrane Ca2+ channels under NaCl stress, which was partially abolished by CAT. These results, taken together, indicate that hydrogen peroxide (H?O?) likely regulates Na+ uptake by activating plasma membrane Ca2+ channels in GCPs. In accordance with this hypothesis, H?O? could mimic extracellular Ca2+ to activate Ca2+ channels and block Na+ influx in guard cells. A single-cell analysis of cytosolic free Ca2+ ([Ca2+](cyt)) using Fluo 3-AM revealed that extracellular Ca2+ induced the accumulation of cytosolic Ca2+ under NaCl stress, but had few effects on the accumulation of cytosolic Ca2+ under non-NaCl conditions. All of these results, together with our previous studies showing that extracellular Ca2+ induced the generation of H?O? in GCPs during NaCl stress, indicate that extracellular Ca2+ alleviates salt stress, likely by activating the H?O?-dependent plasma membrane Ca2+ channels, and the increase in cytosolic Ca2+ appears to block Na+ influx across the plasma membrane in Vicia guard cells, leading to stomatal closure and reduction of water loss.     

Application of diagnostic methods and molecular diagnosis of hemoglobin disorders in Khuzestan province of Iran

BACKGROUND:

The hemoglobinopathies refer to a diverse group of inherited disorders characterized by a reduced synthesis of one or more globin chains (thalassemias) or the synthesis of structurally abnormal hemoglobin (Hb). The thalassemias often coexist with a variety of structural Hb variants giving rise to complex genotypes and an extremely wide spectrum of clinical and hematological phenotypes. Hematological and biochemical investigations and family studies provide essential clues to the different interactions and are fundamental to DNA diagnostics of the Hb disorders. Although DNA diagnostics have made a major impact on our understanding and detection of the hemoglobinopathies, DNA mutation testing should never be considered a shortcut or the test of first choice in the workup of a hemoglobinopathy.

MATERIALS AND METHODS:

A careful three-tier approach involving: (1) Full blood count (2) Special hematological tests, followed by (3) DNA mutation analysis, provides the most effective way in which to detect primary gene mutations as well as gene-gene interactions that can influence the overall phenotype. With the exception of a few rare deletions and rearrangements, the molecular lesions causing hemoglobinopathies are all identifiable by PCR-based techniques. Furthermore, each at-risk ethnic group has its own combination of common Hb variants and thalassemia mutations. In Iran, there are many different forms of α and β thalassemia. Increasingly, different Hb variants are being detected and their effects per se or in combination with the thalassemias, provide additional diagnostic challenges.

RESULTS:

We did step-by-step diagnosis workup in 800 patients with hemoglobinopathies who referred to Research center of Thalassemia and Hemoglobinopathies in Shafa Hospital of Ahwaz Joundishapour University of medical sciences, respectively. We detected 173 patients as iron deficiency anemia (IDA) and 627 individuals as thalassemic patients by use of different indices. We have successfully detected 75% (472/627) of the β-thalassemia mutations by using amplification refractory mutation system (ARMS) technique and 19% (130/627) of the β-thalassemia mutations by using Gap-PCR technique and 6% (25/627) as Hb variants by Hb electrophoresis technique. We did prenatal diagnosis (PND) for 176 couples which had background of thalassemia in first pregnancy. Result of PND diagnosis in the first trimester was 35% (62/176) affected fetus with β-thalassemia major and sickle cell disease that led to termination of the pregnancy.

CONCLUSION:

Almost all hemoglobinopathies can be detected with the current PCR-based assays with the exception of a few rare deletions. However, the molecular diagnostic service is still under development to try and meet the demands of the population it serves. In the short term, the current generation of instruments such as the capillary electrophoresis systems, has greatly simplified DNA sequence analysis.     

Molecular basis of Ca(2)+ activation of the mouse cardiac Ca(2)+ release channel (ryanodine receptor).

Activation of the cardiac ryanodine receptor (RyR2) by Ca(2)+ is an essential step in excitation-contraction coupling in heart muscle. However, little is known about the molecular basis of activation of RyR2 by Ca(2)+. In this study, we investigated the role in Ca(2)+ sensing of the conserved glutamate 3987 located in the predicted transmembrane segment M2 of the mouse RyR2. Single point mutation of this conserved glutamate to alanine (E3987A) reduced markedly the sensitivity of the channel to activation by Ca(2)+, as measured by using single-channel recordings in planar lipid bilayers and by [(3)H]ryanodine binding assay. However, this mutation did not alter the affinity of [(3)H]ryanodine binding and the single-channel conductance. In addition, the E3987A mutant channel was activated by caffeine and ATP, was inhibited by Mg(2)+, and was modified by ryanodine in a fashion similar to that of the wild-type channel. Coexpression of the wild-type and mutant E3987A RyR2 proteins in HEK293 cells produced individual single channels with intermediate sensitivities to activating Ca(2)+. These results are consistent with the view that glutamate 3987 is a major determinant of Ca(2)+ sensitivity to activation of the mouse RyR2 channel, and that Ca(2)+ sensing by RyR2 involves the cooperative action between ryanodine receptor monomers. The results of this study also provide initial insights into the structural and functional properties of the mouse RyR2, which should be useful for studying RyR2 function and regulation in genetically modified mouse models.     

TRPC3-mediated Ca influx contributes to Rac1-mediated production of reactive oxygen species in MLP-deficient mouse hearts

Dilated cardiomyopathy (DCM) is a myocardial disorder that is characterized by dilation and dysfunction of the left ventricle (LV). Accumulating evidence has implicated aberrant Ca²⁺ signaling and oxidative stress in the progression of DCM, but the molecular details are unknown. In the present study, we report that inhibition of the transient receptor potential canonical 3 (TRPC3) channels partially prevents LV dilation and dysfunction in muscle LIM protein-deficient (MLP (−/−)) mice, a murine model of DCM. The expression level of TRPC3 and the activity of Ca²⁺/calmodulin-dependent kinase II (CaMKII) were increased in MLP (−/−) mouse hearts. Acitivity of Rac1, a small GTP-binding protein that participates in NADPH oxidase (Nox) activation, and the production of reactive oxygen species (ROS) were also increased in MLP (−/−) mouse hearts. Treatment with pyrazole-3, a TRPC3 selective inhibitor, strongly suppressed the increased activities of CaMKII and Rac1, as well as ROS production. In contrast, activation of TRPC3 by 1-oleoyl-2-acetyl-sn-glycerol (OAG), or by mechanical stretch, induced ROS production in rat neonatal cardiomyocytes. These results suggest that up-regulation of TRPC3 is responsible for the increase in CaMKII activity and the Nox-mediated ROS production in MLP (−/−) mouse cardiomyocytes, and that inhibition of TRPC3 is an effective therapeutic strategy to prevent the progression of DCM.     

Generation of a neuro-specific microarray reveals novel differentially expressed noncoding RNAs in mouse models for neurodegenerative diseases

We have generated a novel, neuro-specific ncRNA microarray, covering 1472 ncRNA species, to investigate their expression in different mouse models for central nervous system diseases. Thereby, we analyzed ncRNA expression in two mouse models with impaired calcium channel activity, implicated in Epilepsy or Parkinson''s disease, respectively, as well as in a mouse model mimicking pathophysiological aspects of Alzheimer''s disease. We identified well over a hundred differentially expressed ncRNAs, either from known classes of ncRNAs, such as miRNAs or snoRNAs or which represented entirely novel ncRNA species. Several differentially expressed ncRNAs in the calcium channel mouse models were assigned as miRNAs and target genes involved in calcium signaling, thus suggesting feedback regulation of miRNAs by calcium signaling. In the Alzheimer mouse model, we identified two snoRNAs, whose expression was deregulated prior to amyloid plaque formation. Interestingly, the presence of snoRNAs could be detected in cerebral spine fluid samples in humans, thus potentially serving as early diagnostic markers for Alzheimer''s disease. In addition to known ncRNAs species, we also identified 63 differentially expressed, entirely novel ncRNA candidates, located in intronic or intergenic regions of the mouse genome, genomic locations, which previously have been shown to harbor the majority of functional ncRNAs.     

Model of the outer membrane potential generation by the inner membrane of mitochondria.

Voltage-dependent anion channels in the outer mitochondrial membrane are strongly regulated by electrical potential. In this work, one of the possible mechanisms of the outer membrane potential generation is proposed. We suggest that the inner membrane potential may be divided on two resistances in series, the resistance of the contact sites between the inner and outer membranes and the resistance of the voltage-dependent anion channels localized beyond the contacts in the outer membrane. The main principle of the proposed mechanism is illustrated by simplified electric and kinetic models. Computational behavior of the kinetic model shows a restriction of the steady-state metabolite flux through the mitochondrial membranes at relatively high concentration of the external ADP. The flux restriction was caused by a decrease of the voltage across the contact sites and by an increase in the outer membrane potential (up to +60 mV) leading to the closure of the voltage-dependent anion channels localized beyond the contact sites. This mechanism suggests that the outer membrane potential may arrest ATP release through the outer membrane beyond the contact sites, thus tightly coordinating mitochondrial metabolism and aerobic glycolysis in tumor and normal proliferating cells.     

Role of leaflet asymmetry in the permeability of model biological membranes to protons, solutes, and gases.

Bilayer asymmetry in the apical membrane may be important to the barrier function exhibited by epithelia in the stomach, kidney, and bladder. Previously, we showed that reduced fluidity of a single bilayer leaflet reduced water permeability of the bilayer, and in this study we examine the effect of bilayer asymmetry on permeation of nonelectrolytes, gases, and protons. Bilayer asymmetry was induced in dipalmitoylphosphatidylcholine liposomes by rigidifying the outer leaflet with the rare earth metal, praseodymium (Pr3+). Rigidification was demonstrated by fluorescence anisotropy over a range of temperatures from 24 to 50 degrees C. Pr3+-treatment reduced membrane fluidity at temperatures above 40 degrees C (the phase-transition temperature). Increased fluidity exhibited by dipalmitoylphosphatidylcholine liposomes at 40 degrees C occurred at temperatures 1-3 degrees C higher in Pr3+-treated liposomes, and for both control and Pr3+-treated liposomes permeability coefficients were approximately two orders of magnitude higher at 48 degrees than at 24 degrees C. Reduced fluidity of one leaflet correlated with significantly reduced permeabilities to urea, glycerol, formamide, acetamide, and NH3. Proton permeability of dipalmitoylphosphatidylcholine liposomes was only fourfold higher at 48 degrees than at 24 degrees C, indicating a weak dependence on membrane fluidity, and this increase was abolished by Pr3+. CO2 permeability was unaffected by temperature. We conclude: (a) that decreasing membrane fluidity in a single leaflet is sufficient to reduce overall membrane permeability to solutes and NH3, suggesting that leaflets in a bilayer offer independent resistances to permeation, (b) bilayer asymmetry is a mechanism by which barrier epithelia can reduce permeability, and (c) CO(2) permeation through membranes occurs by a mechanism that is not dependent on fluidity.     

Biophysical re-equilibration of Ca2+ fluxes as a simple biologically plausible explanation for complex intracellular Ca2+ release patterns

Physiological regulation of Ca(2+) release from the endoplasmic reticulum (ER) is critical for cell function. Recent direct measurements of free [Ca(2+)] inside the ER ([Ca(2+)](ER)) revealed that [Ca(2+)](ER) itself is a key regulator of ER Ca(2+) handling. However, the role of this new regulatory process in generating various patterns of Ca(2+) release remains to be elucidated in detail. Here, we incorporate the recently quantified experimental correlations between [Ca(2+)](ER) and Ca(2+) movements across the ER membrane into a mathematical model ER Ca(2+) handling. The model reproduces basic experimental dynamics of [Ca(2+)](ER). Although this was not goal in model design, the model also exhibits mechanistically unclear experimental phenomena such as "quantal" Ca(2+) release, and "store charging" by increasing resting cytosolic [Ca(2+)]. While more complex explanations cannot be ruled out, on the basis of our data we propose that "quantal release" and "store charging" could be simple re-equilibration phenomena, predicted by the recently quantified biophysical dynamics of Ca(2+) movements across the ER membrane.     

Ubiquinone (coenzyme Q10) binding sites: low dielectric constant of the gate prevents the escape of the semiquinone

The photosynthetic reaction center (RC) from purple bacteria is frequently used as a model for the interaction of ubiquinones (coenzyme Q) with membrane proteins. Single-turnover flash activation of RC leads to formation of the semiquinone (SQ) of the secondary acceptor quinone after odd flashes and quinol after even flashes. The ubiquinol escapes the binding site in 1 ms, while the SQ does not leave the binding site for at least 5 min. Observed difference between these times suggests a large energetic barrier for the SQ. However, high apparent dielectric constant in the vicinity of the quinone ring (>or=25) results in a relatively small electrostatic energy of SQ stabilization. To resolve this apparent contradiction I suggest that a significant part of the kinetic stabilization of the SQ is achieved by the special topology of the binding site in which quinone can exit the binding site only by moving its headgroup toward the center of the membrane. The large energetic penalty of transferring the charged headgroup to the membrane dielectric can explain the observed kinetic stability of the SQ.