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Biomedical subjects

A A Waheed

Publications and source records attributed to A A Waheed.

15 recordsLinked to original sources
PubMed2003

Concentration of rafts in platelet filopodia correlates with recruitment of c-Src and CD63 to these domains.

The molecular mechanism that causes non-adhesive, discoid platelets to transform into sticky dendritic bodies that form blood clumps is a complex series of events. Recently it has become clear that lipid microdomains--also known as rafts--play a crucial role in this process. We have used a non-cytolytic derivative of perfringolysin-O, a cholesterol binding cytolysin, that binds selectively to cholesterol-rich membrane domains, combined with confocal- and immunoelectron microscopy to visualize cholesterol-raft dynamics during platelet adhesion. In resting platelets cholesterol was uniformly distributed on the cell surface and confined to distinct intracellular compartments (i.e. multivesicular bodies, dense granules, and the internal membranes of alpha-granules). Upon interaction with fibrinogen, cholesterol accumulated at the tips of filopodia and at the leading edge of spreading cells. Stimulation with thrombin receptor activating peptide (TRAP) resulted in a similar redistribution of cholesterol towards filopodia. The adhesion-dependent raft aggregation was accompanied by concentration of the tyrosine kinase c-Src and the tetraspanin CD63 in these domains, whereas glycoprotein Ib (GPIb) was not selectively targeted to the raft clusters. c-Src, the tetraspanin CD63, and GPIb were recovered in biochemically isolated low-density membrane fractions. Disruption of rafts by depleting membrane cholesterol had no effect on platelet shape change but inhibited platelet spreading on fibrinogen and TRAP-induced aggregation. Our results demonstrate that cholesterol rafts in platelets are dynamic entities in the membrane that co-cluster with the tyrosine kinase c-Src and the costimulatory molecule CD63 in specialized domains at the cell surface, thereby providing a possible mechanism in functioning as signaling centres.

Antigens, CD↗
PubMed2001

Scientists turn to journals in English.

Biomedical publications listed in Medline were analyzed based on publisher's location and first author's country of origin. In the present analysis I wished to determine the languages of biomedical publications and the publishers' locations.

Humans↗
PubMed2001

Selective binding of perfringolysin O derivative to cholesterol-rich membrane microdomains (rafts).

There is increasing evidence that sphingolipid- and cholesterol-rich microdomains (rafts) exist in the plasma membrane. Specific proteins assemble in these membrane domains and play a role in signal transduction and many other cellular events. Cholesterol depletion causes disassembly of the raft-associated proteins, suggesting an essential role of cholesterol in the structural maintenance and function of rafts. However, no tool has been available for the detection and monitoring of raft cholesterol in living cells. Here we show that a protease-nicked and biotinylated derivative (BCtheta) of perfringolysin O (theta-toxin) binds selectively to cholesterol-rich microdomains of intact cells, the domains that fulfill the criteria of rafts. We fractionated the homogenates of nontreated and Triton X-100-treated platelets after incubation with BCtheta on a sucrose gradient. BCtheta was predominantly localized in the floating low-density fractions (FLDF) where cholesterol, sphingomyelin, and Src family kinases are enriched. Immunoelectron microscopy demonstrated that BCtheta binds to a subpopulation of vesicles in FLDF. Depletion of 35% cholesterol from platelets with cyclodextrin, which accompanied 76% reduction in cholesterol from FLDF, almost completely abolished BCtheta binding to FLDF. The staining patterns of BCtheta and filipin in human epidermoid carcinoma A431 cells with and without cholesterol depletion suggest that BCtheta binds to specific membrane domains on the cell surface, whereas filipin binding is indiscriminate to cell cholesterol. Furthermore, BCtheta binding does not cause any damage to cell membranes, indicating that BCtheta is a useful probe for the detection of membrane rafts in living cells.

Bacterial Toxins↗
PubMed2000

Mechanism of dye binding in the protein assay using eosin dyes.

Eosin B and eosin Y have been used to estimate micro- and submicrogram quantities of proteins respectively as shown in our previous reports. In the present study we describe the mechanism of eosin binding to proteins. At pH lower than 3.0 the absorbance of unbound dye is greatly reduced. After the dye binds to protein, the absorption maximum of the dye changes from 514 to 530 +/- 5 nm. The absorbance and bathochromatic shift in absorption maximum of the protein-dye complex are proportional to the concentration of protein. The pH of the assay solution does not change due to protein. Arginine, histidine, and lysine (at both acidic and neutral pH) and tryptophan (at acidic pH) residues of a protein bind electrostatically to carboxylic and phenolic groups of the dye to produce a stable water-soluble protein-dye complex. The binding constants of eosin B with poly-L-arginine, poly-L-histidine, poly-L-lysine, and poly-L-tryptophan at pH 1.96 are 0.37, 0.32, 0.33 and 0.33 nmol/nmol of amino acid, respectively. The binding constants of eosin B and eosin Y with bovine serum albumin (BSA) at pH 1.96 are essentially the same, i.e., 0.82 nmol/nmol of reactive amino acid of BSA. The binding constant varies with solution pH so that a wide range of protein concentrations can be estimated. The reason for the higher absorbance of protein-eosin Y complex compared to that of protein-eosin B complex is discussed.

Animals↗
PubMed2000

Estimation of submicrogram quantities of protein using the dye eosin Y.

Eosin B is used to estimate proteins above 1 microg/ml concentration [Waheed AA, Gupta, PD. Anal. Biochem. 1996:233:249-256; Waheed AA, Gupta PD. J. Biochem. Biophys. Meth. 1996;33:187-196]. In the present report we describe a method for estimating submicrogram quantities of proteins using the dye eosin Y. The increase in sensitivity of this assay is approximately two fold under optimal assay condition. The optimum concentration of eosin Y and citric acid for submicrogram assay is 0.01 and 0.05%; (final concentration) respectively. The protein-dye complex formation is completed within 2 min and its absorbance is stable up to 60 main with a variation of +/-4.0%. The interference due to sugars, reducing agents, glycerol and some neutral detergents like Triton X-100, NP-40 and Tween-20 is less than 12% whereas Brij-35, ethanol, acetone and chelators like EGTA and EDTA suppress the absorbance by about 12-18%. However, basic buffers like Tris, urea, CHAPS and NaN, interfere with the formation of the protein-dye complex. The increase in absorbance of protein-eosin Y complex compared to that of protein-eosin B complex is due to the higher extinction co-efficient of eosin Y compared to eosin B.

Animals↗
PubMed1998

Decreased densities of intramembranous particles and cytochemically detectable cholesterol in microvilli of starved rat enterocytes.

The densities of intramembranous particles (IMPs) and of sterol complexes induced by treatment of filipin were studied by freeze-fracture replication of intact intestine and/or isolated brush border membranes (BBM) of well-fed and starved rats. The density of IMPs and filipin-sterol complexes (FSCs) decrease considerably during starvation. Biochemical estimations show a decrease in the levels of cholesterol and proteins with respect to phospholipids during starvation which is in agreement with morphological findings. It is suggested that these changes may play a role in regulating membrane fluidity which in turn affects absorption of nutrients through BBM.

Animals↗
PubMed1998

Lipid and fatty acid composition of brush border membrane of rat intestine during starvation.

Alterations in the lipid and fatty acid composition of brush border membrane (BBM) of small intestine were studied in well-fed, starved, and refed rats. The ratios of cholesterol/phospholipid (mol/mol), sphingomyelin/phosphatidylcholine (mol/mol), protein/lipid (w/w), and free fatty acids (w/w) decreased whereas the total phospholipid (w/w) ratio and the double-bond index increased in BBM of the intestine of the starved rat compared to that of the well-fed rat. Analyses of fatty acids showed higher percentage of stearic and arachidonic acids whereas oleic and linoleic acids decreased under starvation. The acyl chain of starved rat BBM was less ordered compared with that of well-fed rat BBM. On refeeding, these changes were restored to well-fed levels. The change in membrane state under starvation is associated with alterations in the lipid and fatty acid composition of BBM and may be responsible for functional changes that occur under nutritional stress.

Animals↗
PubMed1997

Changes in structural and functional properties of rat intestinal brush border membrane during starvation.

Changes in surface area of microvilli, fluidity of brush border membrane and transport of L-amino acids through intestinal epithelial cells were studied in wellfed and starved (2,4 and 6 days) rats. The surface area of microvilli per unit area of intestinal epithelial cells increased during starvation. Studies with fluoroprobes - pyrene, 1-anilinonaphthalene-8-sulphonate and 1,6-diphenyl-1,3,5-hexatriene, showed increased fluidity of brush border membrane on progressive starvation. Transport of five amino acids representing five different transport systems was studied during starvation in everted intestinal sleeves. Transport of L-proline, glycine and L-glutamic acid which represent imino, glycine and acidic systems respectively increased significantly in Na+-dependent pathway whereas transport of L-lysine representing basic system increased significantly in Na+-independent pathway during starvation.

Amino Acids↗
PubMed1996

Application of an eosin B dye method for estimating a wide range of proteins.

We describe a detailed procedure for estimating a wide range of proteins by the eosin B dye method. At acidic pH, eosin B binds to proteins and absorption of the protein-dye complex at 536-544 nm is proportional to the concentration of the proteins. Inorganic and organic acids are used for the assay, the optimum concentration of acid for each assay differs from acid to acid. This method provides minimal interference with commonly used compounds in protein purification and biomolecules like DNA, RNA, lipids, carbohydrates and minerals which are generally present in biological fluids. This method is applicable for estimating proteins in tissue homogenates, isolated proteins and various biological fluids.

Acids↗
PubMed1992

Effect of starvation on glucose transport and membrane fluidity in rat intestinal epithelial cells.

Studies on the surface area of microvilli (MV), fluidity of brush border membranes (BBM) and D-glucose uptake were carried out in rat intestinal epithelial cells (IEC) during progressive starvation and under re-feed conditions. The surface area of MV, fluidity of BBM and D-glucose transport through IEC membranes showed an increase during starvation when compared to well-fed controls. Re-feeding experiments restored the control values of all the three parameters within a short time. The results showed that the increase in D-glucose transport through IEC membranes during starvation is due to increased surface area of MV and increased fluidity of BBM.

Alkaline Phosphatase↗