Cholesterol organization in membranes at low concentrations: effects of curvature stress and membrane thickness

Cholesterol is often found distributed nonrandomly in domains in biological and model membranes and has been reported to be distributed heterogeneously among various intracellular membranes. Although a large body of literature exists on the organization of cholesterol in plasma membranes or membranes with high cholesterol content, very little is known about organization of cholesterol in membranes containing low amounts of cholesterol. Using a fluorescent cholesterol analog (25-[N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-methyl]amino]-27-norcholesterol, or NBD-cholesterol), we have previously shown that cholesterol may exhibit local organization even at very low concentrations in membranes, which could possibly be attributable to transbilayer tail-to-tail dimers. This is supported by similar observations reported by other groups using cholesterol or dehydroergosterol, a naturally occurring fluorescent cholesterol analog which closely mimics cholesterol. In this paper, we have tested the basic features of cholesterol organization in membranes at low concentrations using spectral features of dehydroergosterol. More importantly, we have investigated the role of membrane surface curvature and thickness on transbilayer dimer arrangement of cholesterol using NBD-cholesterol. We find that dimerization is not favored in membranes with high curvature. However, cholesterol dimers are observed again if the curvature stress is relieved. Further, we have monitored the effect of membrane thickness on the dimerization process. Our results show that the dimerization process is stringently controlled by a narrow window of membrane thickness. Interestingly, this type of local organization of NBD-cholesterol at low concentrations is also observed in sphingomyelin-containing membranes. These results could be significant in membranes that have very low cholesterol content, such as the endoplasmic reticulum and the inner mitochondrial membrane, and in trafficking and sorting of cellular cholesterol.     

SDS Induced Structural Changes in α-Crystallin and It’s Effect on Refolding

-Crystallin, a major eye lens protein and a key member of the small heat shock protein family, acts like a chaperone by preventing aggregation of substrate proteins. One of the hallmarks of most small heat shock proteins is their existence as a large oligomer, the role of which in its function is not understood at present. We have studied the role of the oligomer in the stability of its structure against SDS induced destabilization by CD measurements. -Crystallin from bovine source as well as recombinant preparation was used for this purpose. As SDS concentration was gradually increased, the -sheet structure was diminished followed by concomitant increase in the -helical structure. The quaternary structural changes in presence of SDS were also monitored by light scattering, polarization and anisotropy measurements. It was found that the breakdown of the oligomeric structure was nearly complete above 1 mM SDS concentration. The results were compared with that of a monomeric -crystallin, which is also a major -sheet protein like -crystallin. When -crystallin was first converted into monomeric random coil structure in presence of 6 M urea and allowed to refold in SDS solution, amount of -helix was more than that incubated directly in the same concentration of SDS. The results show that -crystallin attains extra structural stability against external stress due to its oligomeric structure. The implication for the extra stability is discussed in reference to its function as molecular chaperone.     

A proteomic approach to understanding the development of multidrug-resistant<Emphasis Type="Italic"> Candida albicans</Emphasis> strains

Resistance of the pathogenic yeast Candida albicans to the antifungal agent fluconazole is often caused by the overexpression of genes that encode multidrug efflux pumps (CDR1, CDR2, or MDR1). We have undertaken a proteomic approach to gain further insight into the regulatory network controlling efflux pump expression and drug resistance in C. albicans. Three pairs of matched fluconazole-susceptible and resistant clinical C. albicans isolates, in which drug resistance correlated with stable activation of MDR1 or CDR1/2, were analyzed for differences in their protein expression profiles. In two independent, MDR1-overexpressing, strains, additional up-regulated proteins were identified, which are encoded by the YPR127 gene and several members of the IFD (YPL088) gene family. All are putative aldo-keto reductases of unknown function. These proteins were not up-regulated in a fluconazole-resistant strain that overexpressed CDR1 and CDR2 but not MDR1, indicating that expression of the various efflux pumps of C. albicans is controlled by different regulatory networks. To investigate the possible role of YPR127 in the resistance phenotype of the clinical isolates, we constitutively overexpressed the gene in a C. albicans laboratory strain. In addition, the gene was deleted in a C. albicans laboratory strain and in one of the drug-resistant clinical isolates in which it was overexpressed. Neither forced overexpression nor deletion of YPR127 affected the susceptibility of the strains to drugs and other toxic substances, suggesting that the regulatory networks which control the expression of efflux pumps in C. albicans also control genes involved in cellular functions not related to drug resistance.Communicated by D. Y. Thomas     

Combination of ascorbate and alpha-tocopherol as a preventive therapy against structural and functional defects of erythrocytes in visceral leishmaniasis

The redox unbalance in erythrocytes has been found to contribute significantly in the development of anemia in visceral leishmaniasis (VL). The present study revealed enhanced production of reactive oxygen species (ROS) and gradual depletion of alpha-tocopherol and ascorbate in the erythrocytes of infected animals. The response of erythrocytes to chronic treatment with antioxidants was studied in hamsters during leishmanial infection. Treatment with a combination of alpha-tocopherol and ascorbate proved to be the most effective preventive for the proteolytic degradation of erythrocyte membrane. Erythrocytes from infected animals were thermally more sensitive compared to the control ones. Combination of both antioxidants was most successful in resisting heat induced structural defects in the cells. Cross-linking of membrane proteins subsequent to oxidative damage in the red cells was accompanied by the formation of high molecular weight protein band at the top of the resolving gel in the presence of the cross-linking agent dimethyladepimidate (DMA). Marked inhibition of cross-linking was observed with combination of both antioxidants. Treatment with alpha-tocopherol and ascorbate together could withstand osmotic lysis of erythrocytes in the infected animals very efficiently. Decreased hemoglobin (Hb) level was successfully replenished and was coupled with significant increase in the life span of red cells after treating the animals with both antioxidants. Results indicate better efficacy of the combination therapy with alpha-tocopherol and ascorbate in protecting the erythrocytes from structural and functional damages during leishmanial infection.     

Ebselen inhibits NO-induced apoptosis of differentiated PC12 cells via inhibition of ASK1-p38 MAPK-p53 and JNK signaling and activation of p44/42 MAPK and Bcl-2

Ebselen, a selenium-containing heterocyclic compound, prevents ischemia-induced cell death. However, the molecular mechanism through which ebselen exerts its cytoprotective effect remains to be elucidated. Using sodium nitroprusside (SNP) as a nitric oxide (NO) donor, we show here that ebselen potently inhibits NO-induced apoptosis of differentiated PC12 cells. This was associated with inhibition of NO-induced phosphatidyl Serine exposure, cytochrome c release, and caspase-3 activation by ebselen. Analysis of key apoptotic regulators during NO-induced apoptosis of differentiated PC12 cells showed that ebselen blocks the activation of the apoptosis signaling-regulating kinase 1 (ASK1), and inhibits phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal protein kinase (JNK). Moreover, ebselen inhibits NO-induced p53 phosphorylation at Ser15 and c-Jun phosphorylation at Ser63 and Ser73. It appears that inhibition of p38 MAPK and p53 phosphorylation by ebselen occurs via a thiol-redox-dependent mechanism. Interestingly, ebselen also activates p44/42 MAPK, and inhibits the downregulation of the antiapoptotic protein Bcl-2 in SNP-treated PC12 cells. Together, these findings suggest that ebselen protects neuronal cells from NO cytotoxicity by reciprocally regulating the apoptotic and antiapoptotic signaling cascades.     

Mechanism and stoichiometry of interaction of DnaG primase with DnaB helicase of Escherichia coli in RNA primer synthesis

Initiation and synthesis of RNA primers in the lagging strand of the replication fork in Escherichia coli requires the replicative DnaB helicase and the DNA primase, the DnaG gene product. In addition, the physical interaction between these two replication enzymes appears to play a role in the initiation of chromosomal DNA replication. In vitro, DnaB helicase stimulates primase to synthesize primers on single-stranded (ss) oligonucleotide templates. Earlier studies hypothesized that multiple primase molecules interact with each DnaB hexamer and single-stranded DNA. We have examined this hypothesis and determined the exact stoichiometry of primase to DnaB hexamer. We have also demonstrated that ssDNA binding activity of the DnaB helicase is necessary for directing the primase to the initiator trinucleotide and synthesis of 11-20-nucleotide long primers. Although, association of these two enzymes determines the extent and rate of synthesis of the RNA primers in vitro, direct evidence of the formation of primase-DnaB complex has remained elusive in E. coli due to the transient nature of their interaction. Therefore, we stabilized this complex using a chemical cross-linker and carried out a stoichiometric analysis of this complex by gel filtration. This allowed us to demonstrate that the primase-helicase complex of E. coli is comprised of three molecules of primase bound to one DnaB hexamer. Fluorescence anisotropy studies of the interaction of DnaB with primase, labeled with the fluorescent probe Ru(bipy)3, and Scatchard analysis further supported this conclusion. The addition of DnaC protein, leading to the formation of the DnaB-DnaC complex, to the simple priming system resulted in the synthesis of shorter primers. Therefore, interactions of the DnaB-primase complex with other replication factors might be critical for determining the physiological length of the RNA primers in vivo and the overall kinetics of primer synthesis.     

Conformational dynamics of DnaB helicase upon DNA and nucleotide binding: analysis by intrinsic tryptophan fluorescence quenching

DnaB helicase of E. coli unwinds duplex DNA in the replication fork using the energy of ATP hydrolysis. We have analyzed structural and conformational changes in the DnaB protein in various nucleotides and DNA bound intermediate states by fluorescence quenching analysis of intrinsic fluorescence of native tryptophan (Trp) residues in DnaB. Fluorescence quenching analysis indicated that Trp48 in domain alpha is in a hydrophobic environment and resistant to fluorescence quenchers such as potassium iodide (KI). In domain beta, Trp294 was found to be in a partially hydrophobic environment, whereas Trp456 in domain gamma appeared to be in the least hydrophobic environment. Binding of oligonucleotides to DnaB helicase resulted in a significant attenuation of the fluorescence quenching profile, indicating a change in conformation. ATPgammaS or ATP binding appeared to lead to a conformation in which Trp residues had a higher degree of solvent exposure and fluorescence quenching. However, the most dramatic increase of Trp fluorescence quenching was observed with ADP binding with a possible conformational relaxation. Site-specific Trp --> Cys mutants of DnaB helicase demonstrated that conformational change upon ADP binding could be attributed exclusively to a conformational transition in the alpha domain leading to an increase in the solvent exposure of Trp48. However, formation of DnaB.ATPgammaS.DNA ternary complex led to a conformation with a fluorescence quenching profile similar to that observed with DnaB alone. The DnaB.ADP.DNA ternary complex produced a quenching curve similar to that of DnaB.ADP complex pointing to a change in conformation due to ATP hydrolysis. There are at least four identifiable structural/conformational states of DnaB helicase that are likely important in the helicase activity. The noncatalytic alpha domain in the N-terminus appeared to undergo the most significant conformational changes during nucleotide binding and hydrolysis. This is the first reported elucidation of the putative role of domain alpha, which is essential for DNA helicase action. We have correlated these results with partial structural models of alpha, beta, and gamma domains     

Protection of adrenocortical activity by dietary casein in ether anaesthetized rats

Adrenal delta5-3beta-hydroxysteroid dehydrogenase (delta5-3beta-HSD) activity and serum corticosterone level were significantly higher in rats fed with 5% casein or 4% albumin diets after 1 hr of ether anaesthetic stress as compared to the controls, 5% casein and 20% casein (equivalent to 4% albumin) respectively. Ether anaesthesia to 20% casein fed rats caused no change in adrenal delta5-3beta-HSD activity and serum corticosterone level when compared with controls fed 20% casein diet. The results suggest that high milk protein diet may prevent acute stress effects by protecting adrenocortical activity. The present investigation opens up a new area of management of stress.     

A novel antioxidant and antiapoptotic role of omeprazole to block gastric ulcer through scavenging of hydroxyl radical

The mechanism of the antiulcer effect of omeprazole was studied placing emphasis on its role to block oxidative damage and apoptosis during ulceration. Dose-response studies on gastroprotection in stress and indomethacin-induced ulcer and inhibition of pylorus ligation-induced acid secretion indicate that omeprazole significantly blocks gastric lesions at lower dose (2.5 mg/kg) without inhibiting acid secretion, suggesting an independent mechanism for its antiulcer effect. Time course studies on gastroprotection and acid reduction also indicate that omeprazole almost completely blocks lesions at 1 h when acid inhibition is partial. The severity of lesions correlates well with the increased level of endogenous hydroxyl radical (*OH), which when scavenged by dimethyl sulfoxide causes around 90% reduction of the lesions, indicating that *OH plays a major role in gastric damage. Omeprazole blocks stress-induced increased generation of *OH and associated lipid peroxidation and protein oxidation, indicating that its antioxidant role plays a major part in preventing oxidative damage. Omeprazole also prevents stress-induced DNA fragmentation, suggesting its antiapoptotic role to block cell death during ulceration. The oxidative damage of DNA by *OH generated in vitro is also protected by omeprazole or its analogue, lansoprazole. Lansoprazole when incubated in a *OH-generating system scavenges *OH to produce four oxidation products of which the major one in mass spectroscopy shows a molecular ion peak at m/z 385, which is 16 mass units higher than that of lansoprazole (m/z 369). The product shows no additional aromatic proton signal for aromatic hydroxylation in (1)H NMR. The product absorbing at 278 nm shows no alkaline shift for phenols, thereby excluding the formation of hydroxylansoprazole. The product is assigned to lansoprazole sulfone formed by the addition of one oxygen atom at the sulfur center following attack by the *OH. Thus, omeprazole plays a significant role in gastroprotection by acting as a potent antioxidant and antiapoptotic molecule.