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S Mall

Publications and source records attributed to S Mall.

12 recordsLinked to original sources
PubMed2001

Self-association of model transmembrane alpha-helices is modulated by lipid structure.

We have developed a fluorescence quenching method using peptides containing 3,5-dibromotryrosine to measure oligomerization of model transmembrane alpha-helices in lipid bilayers. Peptides of the type Ac-LysLysGlyLeu(m)XLeu(n)LysLysAla-amide where X is tryptophan or 3,5-dibromotyrosine were found to form heterodimers in bilayers of phosphatidylcholine in the liquid-crystalline phase. The free energy of dimer formation changed little with increasing number of Leu residues from 16 to 22 but increased with increasing phospholipid fatty acyl chain length, with a slope of about 0.5 kJ mol(-1) per fatty acyl chain carbon. Peptides were excluded from lipid in the gel phase, resulting in increased levels of oligomerization. Addition of cholesterol to form the liquid-ordered state led to increased dimerization but without phase separation. The presence of phosphatidylethanolamine had little effect on dimerization.

Amino Acid Sequence↗
PubMed2001

Fabrication and characterization of microwell array chemical sensors.

Microwell arrays have been fabricated on the distal face of coherent fiber-optic bundles. A typical microwell array comprises approximately 3,000 individual optical fibers that were etched chemically. Individual microwells were 1 to 14-microm deep with approximately 22-microm widths and were filled partially with a chemical sensing (polymer + dye) layer to produce a microwell array sensor (MWAS). MWASs were fabricated using a technically expedient, photoinitiated polymerization reaction whereby a approximately 2 to 10-microm thick pH-sensitive or O2-sensitive sensing layer was immobilized inside each microwell. The pH-sensing layer comprised fluorescein isothiocyanate-dextran conjugate immobilized in a photopolymerizable poly(vinyl alcohol) membrane. The O2-sensing layer comprised a ruthenium metal complex entrapped in a gas-permeable photopolymerizable siloxane membrane. pH and PO2 were quantitated by acquiring luminescence images using an epifluorescence microscope/charge-coupled device imaging system. The pH-sensitive MWAS displayed a pKa of approximately 6.4 and a response time of approximately 2.5 s. The O2-sensitive MWAS behaved according to a nonlinear Stern-Volmer model with a maximum I0/I of approximately 4 and a response time of approximately 2.5 s. MWASs are advantageous in that suitably sized samples such as single biological cells can be co-localized with the sensing matrix in individual microwells.

Journal Article↗
PubMed2000

Effects of aromatic residues at the ends of transmembrane alpha-helices on helix interactions with lipid bilayers.

We have studied the effects of aromatic residues at the ends of peptides of the type Ac-KKGL(n)()WL(m)()KKA-amide on their interactions with lipid bilayers as a function of lipid fatty acyl chain length, physical phase, and charge. Peptide Ac-KKGFL(6)WL(8)FKKA-amide (F(2)L(14)) incorporated into bilayers of phosphatidylcholines containing monounsaturated fatty acyl chains of lengths C14-C24 at a peptide:lipid molar ratio of 1:100 in contrast to Ac-KKGL(7)WL(9)KKA-amide (L(16)) which did not incorporate at all into dierucoylphosphatidylcholine [di(C24:1)PC]; Ac-KKGYL(6)WL(8)YKKA-amide (Y(2)L(14)) incorporated partly into di(C24:1)PC. Lipid-binding constants relative to that for dioleoylphosphatidylcholine (C18:1)PC were obtained using a fluorescence quenching method. For Y(2)L(14) and F(2)L(14), relative lipid-binding constants increased with increasing fatty acyl chain length from C14 to C24; strongest binding did not occur at the point where the hydrophobic length of the peptide equalled the hydrophobic thickness of the bilayer. For Ac-KKGYL(9)WL(11)YKKA-amide (Y(2)L(20)), increasing chain length from C18 to C24 had little effect on relative binding constants. Anionic phospholipids bound more strongly than zwitterionic phospholipids to Y(2)L(14) and Y(2)L(20) but effects of charge were relatively small. In two phase (gel and liquid crystalline) mixtures, all the peptides partitioned more strongly into liquid crystalline than gel phase; effects were independent of the structure of the peptide or of the lipid (dipalmitoylphosphatidylcholine or bovine brain sphingomyelin). Addition of cholesterol had little effect on incorporation of the peptides into lipid bilayers. It is concluded that the presence of aromatic residues at the ends of transmembrane alpha-helices effectively buffers them against changes in bilayer thickness caused either by an increase in the chain length of the phospholipid or by the presence of cholesterol.

Cholesterol↗
PubMed1999

Anionic phospholipids decrease the rate of slippage on the Ca(2+)-ATPase of sarcoplasmic reticulum.

Accumulation of Ca(2+) by the Ca(2+)-ATPase of skeletal-muscle sarcoplasmic reticulum has been measured in reconstituted, sealed vesicles as a function of lipid composition. Measurements were performed in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to eliminate any effects of H(+) transport; in the presence of FCCP, addition of valinomycin had no effect on the level or rate of accumulation of Ca(2+) showing that, in the presence of FCCP, no electrical potential built up across the membrane. Levels of accumulation were low when the phospholipid was dioleoylphosphatidylcholine (DOPC), even though DOPC supports high ATPase activity. Inclusion of 10 mol% anionic phospholipid [dioleoylphosphatidic acid (DOPA) or dioleoylphosphatidylserine (DOPS)] led to higher levels of accumulation of Ca(2+), 10 mol% being the optimum concentration. Cardiolipin or phosphatidylinositol 4-phosphate were more effective than DOPA or DOPS in increasing accumulation of Ca(2+). Effects of anionic phospholipids were seen in the presence of an ATP-regenerating system to remove ADP, and in the presence of phosphate within the reconstituted vesicles to precipitate calcium phosphate. Rates of passive leak of Ca(2+) from the reconstituted vesicles were slow. The Ca(2+)-accumulation process was simulated assuming either simple passive leak of Ca(2+) from the vesicles or assuming slippage on the ATPase, a process in which the phosphorylated intermediate of the ATPase releases bound Ca(2+) on the cytoplasmic rather than the lumenal side of the membrane. The experimental data fitted to a slippage model, with anionic phospholipids decreasing the rate of slippage.

Adenosine Triphosphate↗
PubMed1998

Interaction of phosphatidic acid and phosphatidylserine with the Ca2+-ATPase of sarcoplasmic reticulum and the mechanism of inhibition.

The sarcoplasmic reticulum of skeletal muscle contains anionic phospholipids as well as the zwitterionic phosphatidylcholine and phosphatidylethanolamine. Here we study the effects of anionic phospholipids on the activity of the Ca2+-ATPase purified from the membrane. Reconstitution of the Ca2+-ATPase into dioleoylphosphatidylserine [di(C18:1)PS] or dioleoylphosphatidic acid [di(C18:1)PA] leads to a decrease in ATPase activity. Measurements of the quenching of the tryptophan fluorescence of the ATPase by brominated phospholipids give a relative binding constant for the anionic lipids compared with dioleoylphosphatidylcholine close to 1 and suggest that phosphatidic acid only binds to the ATPase at the bulk lipid sites around the ATPase. Addition of di(C18:1)PS or di(C18:1)PA to the ATPase in the short-chain dimyristoleoylphosphatidylcholine [di(C14:1)PC] reverse the effects of the short-chain lipid on ATPase activity and on Ca2+ binding, as revealed by the response of tryptophan fluorescence intensity to Ca2+ binding. It is concluded that the lipid headgroup and lipid fatty acyl chains have separate effects on the function of the ATPase. The anionic phospholipids have no significant effect on Ca2+ binding to the ATPase; the level of Ca2+ binding to the ATPase, the affinity of binding and the rate of dissociation of Ca2+ are unchanged by reconstitution into di(C18:1)PA. The major effect of the anionic lipids is a reduction in the maximal level of binding of MgATP. This is attributed to the formation of oligomers of the Ca2+-ATPase, in which only one molecule of the ATPase can bind MgATP dimers in di(C18:1)PS and trimers or tetramers in di(C18:1)PA. The rates of phosphorylation and dephosphorylation for the proportion of the ATPase still able to bind ATP are unaffected by reconstitution. Larger changes were observed in the level of phosphorylation of the ATPase by Pi, which became very low in the anionic phospholipids. The fluorescence response to Mg2+ for the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin was also changed in di(C18:1)PS and di(C18:1)PA, so that effects of Mg2+ became comparable with those seen on phosphorylation for the unreconstituted ATPase. The anionic phospholipids could induce a conformational change in the ATPase on binding Mg2+ equivalent to that normally induced by phosphorylation or by binding inhibitors such as thapsigargin.

Adenosine Triphosphate↗
PubMed1998

Lipid-protein interactions in the membrane: studies with model peptides.

We have used fluorescence quenching of tryptophan-containing trans-membrane peptides by bromine-containing phospholipids to study the specificity of peptide-lipid interactions. We have synthesized peptides Ac-K2GLm WLnK2A-amide where m = 7 and n = 9 (L16) and m = 10 and n = 12 (L22). Binding constants of L22 for dioleoylphosphatidylserine [di(C18 : 1)PS] or dioleoylphosphatidic acid [di(C18 : 1)PA] relative to dieoleoylphosphatidylcholine [di(C18 : 1)PC] were close to 1. However, for L16, whilst the bulk of the di(C18 : 1)PA molecules bound with a binding constant relative to di(C18 : 1)PC close to 1, a small number of di(C18 : 1)PA molecules bound much more strongly. Assuming just one high affinity binding site on L16 for anionic lipid, the affinity of the site for di(C18 : 1)PS was calculated to be ca. 8 times that for di (C18 : 1)PC. The relative binding constant was little affected by ionic strength and close contact between the anionic headgroup of di(C18 : 1)PS and a lysine residue on the peptide was suggested. The relative binding constant for di(C18 : 1)PS at this high affinity site was less than for di(C18 : 1)PA. Cholesterol interacts with L22 with an affinity about 0.7 of that of di(C18 : 1)PC. The structure of the peptide itself is important. The peptide Ac-KKGYL6WL8YKKA-amide (Y2L14) incorporated into bilayers of dinervonylphosphatidylcholine [di(C24 : 1)PC] whereas L16 did not incorporate into this lipid. It is suggested that thinning of a lipid bilayer around a peptide to give optimal hydrophobic matching is less energetically unfavourable when a Tyr residue is located in the lipid/water interfacial region.

Cell Membrane↗
PubMed1997

Expression of the Wilms' tumour gene WT1 in the developing human and in paediatric renal tumours: an immunohistochemical study.

AIMS: The Wilms' tumour gene (WT1) product is expressed during the development of the urogenital system. This study was undertaken to evaluate four anti-WT1 antibodies and use the most specific one to examine the expression of WT1 in formalin fixed, paraffin wax embedded tissues from human embryos, fetuses, and paediatric renal neoplasms. METHODS: The antibodies were assessed on paraffin sections of fetal kidney and by western blotting. Immunohistochemical techniques were optimised and performed on a range of embryonic, fetal, and infant tissues from 35 days post-conception to three months of age, and on a selection of paediatric renal neoplasms. RESULTS: The antibodies tested were found to vary in their specificity. Anomalous expression in smooth muscle was seen with one batch of a commercial polyclonal antibody. WT1 protein was detected in both the metanephros and the mesonephros, the spleen, the gonads, and in the peritoneal mesothelium in fetuses. WT1 was expressed in nuclei and was strongest in the podocytes of fetal kidney. The podocytes of infant glomeruli were also positive. There was focal positive staining in Wilms' tumours, nephrogenic rests, and in a cystic partially differentiated nephroblastoma. Staining of nuclei was seen in one of two rhabdoid tumours of the kidney. No positive staining was seen in other renal tumours. CONCLUSIONS: WT1 is detected readily in formalin fixed material. There were differences in specificity between batches of the polyclonal antibodies used. The distribution of the WT1 gene product in tissues and tumours reflected previous findings with in situ hybridisation studies of WT1 mRNA.

Antibodies↗
PubMed1996

Dissolution behaviour of sulphonamides into sodium dodecyl sulfate micelles: a thermodynamic approach.

The surface energies of four sulfonamides have been assessed from contact angle data, using the Lewis acid-base approach. From these data the free energy of adhesion between the drugs and sodium dodecyl sulfate (SDS) head groups and tails has been calculated. The most favored interaction was for adhesion to the SDS tails, rather than the head groups. The initial rotating disk dissolution rate (hereafter termed dissolution rate) of drug compacts has been measured in water and water with SDS micelles at a range of temperatures. The thermodynamic parameters of activation have been calculated from the rate data. Linear relationships exist between the enthalpy of transfer between water and SDS micelles and the free energy of adhesion between the drugs and both SDS head groups and SDS tails. The most nonpolar drugs had the most favored free energy of adhesion and the most favored enthalpy of transfer. The most polar drug had a disfavoured free energy of adhesion to the SDS head and a disfavoured enthalpy of transfer. This response demonstrates that the most important barrier to the passage from the aqueous fluid to the hydrophobic core of the micelle is the monopolar repulsion between the polar forces of the drug and head group surface energies. This provides a new insight into a possible mechanism of solubilization and offers the prospect of understanding even more complex partitioning behavior.

Chemical Phenomena↗
PubMed1994

Phacolytic glaucoma--its treatment by planned extracapsular cataract extraction with posterior chamber intraocular lens implantation.

Phacolytic glaucoma has traditionally been treated with intracapsular lens extraction to avoid any anaphylaxis. Various mechanisms have been described for the rise of intraocular pressure in these cases. The present study was undertaken to evaluate the response of extracapsular cataract extraction (ECCE) with posterior chamber intraocular lens implantation (PC IOL) in five cases of phacolytic glaucoma that occurred between March 1989 and August 1990. A planned extracapsular cataract extraction with can-opener capsulectomy was done in all the cases with placement of a sulcus-fixated modified J-loop Sinskey design intraocular lens. With a mean follow-up period of two years, all patients (100%) maintained a normal postoperative intraocular pressure of less than 20 mm Hg without any additional medical therapy. The final best-corrected visual acuity in 4 cases (80%) was 6/12 or better, while in one case it was 6/24 due to a senile maculopathy. These results show that ECCE with PC IOL implantation is a safe and efficacious method of visual rehabilitation in cases of phacolytic glaucoma.

Aged↗