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Biomedical subjects

C M Gupta

Publications and source records attributed to C M Gupta.

At least 73 records · Page 4Linked to original sources
PubMed1986

Antibody-mediated targeting of liposomes to erythrocytes in whole blood.

F(ab')2 fragments derived from anti-rat erythrocyte antibody or normal rabbit serum IgG were covalently attached to the surface of liposomes consisting of equimolar amounts of egg phosphatidylcholine and cholesterol. These liposomes were interacted with rat, monkey or mouse blood, and their binding to both red and white blood cells was determined. Results of these studies show that coupling of liposomes to anti-rat erythrocyte F(ab')2 considerably enhances their binding to erythrocytes in rat blood. However, no such increase in the binding was observed with rat leukocytes or monkey and mouse erythrocytes. Besides, the interactions between the liposomes and target cells did not affect the permeability properties of the liposome bilayer. These observations indicate that liposomes coupled to cell-specific antibodies may serve as highly useful carriers for homing of drugs/enzymes to specific cells in biophase.

Animals↗
PubMed1985

Transbilayer phosphatidylcholine distributions in small unilamellar sphingomyelin-phosphatidylcholine vesicles: effect of altered polar head group.

The effect of the altered polar head group of phosphatidylcholine (PC) on its transbilayer distributions in small unilamellar vesicles containing sphingomyelin (SM) was ascertained with phospholipase A2 as the external membrane probe. These vesicles were formed by sonication and fractionated by centrifugation. The vesicle size was determined by gel-permeation chromatography and solute entrapment. Experiments were done to confirm that phospholipase A2 treatments did not induce fusion, lyse the vesicles, or cause PC to migrate across the vesicle bilayer. The complete degradation of external PC in intact vesicles was assured by carrying out the enzyme reactions in the absence as well as in the presence of 9.2 X 10(-5) M bovine serum albumin. In small vesicles comprised of SM and 30 mol % 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), DPPC preferentially distributed in the inner monolayer. This preference of DPPC in these vesicles disappeared upon introducing one C2H5 group at the carbon atom adjacent to the quaternary ammonium residue in its polar head group and was reversed when the C2H5 group was replaced by C6H5 and C6H5CH2 substituents or when the P-N distance was increased. These results indicate that the effective polar head-group volume is an important factor in determining the phospholipid distributions across the small vesicle bilayer.

Lipid Bilayers↗
PubMed1985

Phase behavior of carbamyloxyphosphatidylcholine, a sphingolipid analogue.

The phase behavior of two series of phosphatidylcholines (PC) possessing carbamyloxy-linked fatty acids has been studied by differential scanning calorimetry. These non-natural phosphatidylcholines are of interest because they possess the capability of forming interlipid hydrogen bonds via the carbamyloxy function and as such can serve as a model for sphingolipids, the phase behavior of which is thought to be dominated by interlipid hydrogen bonding. Furthermore, carbamyloxyphosphatidylcholines form unusually stable liposomes, and thus have potential in drug delivery. Carbamyloxyphosphatidylcholines of the type di-(CnH2n+1NHCOO)-PC, where n = 13, 15, or 17, exhibit metastable polymorphism. Cooling from the liquid crystalline state results in formation of a metastable low-temperature polymorph I, which must transform into a stable low-temperature polymorph II before the liquid crystalline state can be reached again. Carbamyloxyphosphatidylcholines of the type 1-C16:0-2-(CnH2n+1NHCOO)-PC exhibit similar metastable polymorphism. This metastability is similar to that exhibited by certain cerebrosides and sphingomyelins and indicates that the sphingosine backbone is not a prerequisite for this type of metastability. Furthermore, the carbamyloxy group is reversed in orientation compared with the amide of sphingolipids (-NHCO- versus -CONH-), suggesting that the intermolecular hydrogen bonding potential, rather than some highly specific steric or conformational constraint, is responsible for the observed metastability of sphingolipids.

Calorimetry, Differential Scanning↗
PubMed1984

Specific interactions of liposomes with PMN leukocytes upon incorporating tuftsin in their bilayers.

Incorporation of tuftsin derivatives, Thr-Lys-Pro-Arg-NH-C18H37 or Thr-Lys-Pro-Arg-NH-(CH2)2-NH-COC15H31, into an egg phosphatidylcholine/cholesterol liposome bilayer led to significantly enhanced binding of the liposomes to PMN leukocytes at 37 degrees C but not at 0 degree C. Under identical conditions, no such enhanced binding of the liposomes was observed with erythrocytes and lymphocytes. These results demonstrate that grafting of tuftsin on the liposome bilayer enables the liposome to recognize specifically the PMN leukocytes and to deliver its contents to these cells.

Cholesterol↗
PubMed1984

Transbilayer distributions of red cell membrane phospholipids in unilamellar vesicles.

The phospholipid organization in unilamellar vesicles comprised of various purified phospholipid components of monkey erythrocyte membrane was ascertained using phospholipase A2 and trinitrobenzenesulfonic acid as external membrane probes. The vesicles were formed by sonication or detergent dialysis and fractionated by centrifugation or gel permeation chromatography. Experiments were done to confirm that the phospholipase A2 treatments did not cause lysis or induce fusion of the vesicles. This enzyme hydrolysed only the glycerophospholipids in the outer surface of the vesicles. The amounts of the external phospholipids determined by this enzymatic method were verified using the chemical probe, trinitrobenzenesulfonic acid. The choline-containing phospholipids and phosphatidylethanolamine localized randomly in the two surfaces of sonicated vesicles (outer diameter, about 30 nm), whereas phosphatidylserine preferentially distributed in the inner monolayer. This phosphatidylserine asymmetry virtually disappeared in detergent dialysed vesicles (outer diameter, about 45 nm). Furthermore, inclusion of cholesterol in both the types of vesicles resulted in more random glycerophospholipid distributions across the plane of vesicles bilayer, presumably due to the cholesterol-induced increases in the size of vesicles. These results demonstrate that the transbilayer distribution of erythrocyte membrane phospholipids in unilamellar vesicles are controlled mainly by the surface curvature rather than by interlipid interactions, and therefore suggest that phospholipid-phospholipid and phospholipid-cholesterol interactions should not play any significant role in determining the membrane phospholipid asymmetry in red cells. It is proposed that this asymmetry primarily originates from differential bindings of phospholipids with membrane proteins in the two leaflets of the membrane bilayer.

Animals↗
PubMed1984

Cytogenetic studies of early myeloid progenitor compartments in Ph1-positive chronic myeloid leukaemia (CML). I. Persistence of Ph1-negative committed progenitors that are suppressed from differentiating in vivo.

We have cytogenetically analysed individual haemopoietic colonies to investigate the level and extent of normal stem cell suppression that occurs in patients with Philadelphia chromosome (Ph1)-positive CML. Seventeen patients were studied at diagnosis prior to the initiation of chemotherapy and five of these were studied again 1-16 months later. Another nine patients were studied for the first time 2-96 months after diagnosis and initiation of chemotherapy. No chromosomally normal metaphases were found in either direct marrow preparations or in haemopoietic colonies obtained from simultaneous assays of marrow and/or blood samples from 20 of the 26 patients studied. In the other six, chromosomally normal haemopoietic progenitors (BFU-E, CFU-C and CFU-G/E) were readily demonstrable even though in five of these patients all dividing cells in the bone marrow appeared to belong to the Ph1-positive clone at the time of study. These results indicate that the suppressive effects of clonal expansion on normal haemopoiesis are more pronounced, and apparent sooner, in the more differentiated compartments. In addition, they support the view that the original population of normal stem cells does not disappear rapidly, although their numbers may be diluted to undetectable levels depending upon the extent of clonal expansion at the stem cell level by the time of diagnosis.

Adolescent↗
PubMed1984

Cytogenetic studies of early myeloid progenitor compartments in Ph1-positive chronic myeloid leukemia. II. Long-term culture reveals the persistence of Ph1-negative progenitors in treated as well as newly diagnosed patients.

We recently showed that long-term marrow cultures can be used to demonstrate the presence of Philadelphia (Ph1) negative progenitors in patients with newly diagnosed Ph1-positive chronic myeloid leukemia (CML). We now report results for 6 chronic phase patients studied 5-83 mo postdiagnosis and an additional 3 newly diagnosed patients. Marrow metaphases were exclusively Ph1-positive. Clonogenic assays revealed a minor population of Ph1-negative progenitors in 3 cases (1 treated, 2 untreated). Long-term marrow culture adherent layers contained Ph1-negative progenitors in 6 cases (3 treated, 3 untreated). Whenever this occurred, the Ph1-negative population had become the only one detectable within 3-4 wk, and this was always associated with a rapid decline of the Ph1-positive population. For 2 of the 3 cases where Ph1-negative progenitors were not detected, there was a similar rapid decline in the Ph1-positive population in culture. In the other case, Ph1-positive progenitors were maintained at levels typically seen in normal long-term marrow cultures. These results suggest that chromosomally normal stem cells may persist for a considerable period in the marrow of some, but perhaps not all, patients with CML, even in the face of maintenance chemotherapy. In addition, they provide new evidence of heterogeneity in this disease, as shown by the variable ability of Ph1-positive progenitor populations to be maintained in vitro.

Adult↗
PubMed1983

The membrane-embedded segment of cytochrome b5 as studied by cross-linking with photoactivatable phospholipids.

Vesicles were prepared from a 9:1 (mole/mol) mixture of dipalmitoyl phosphatidylcholine and the radioactively labeled phospholipids, 1-palmitoyl-2-omega-(m-diazirinophenoxy)undecanoyl-sn-glycero-3-phosphocholine (PC-I) or 1-palmitoyl-2-omega-(2-diazo-3,3,3-trifluropropionyloxy)lauroyl-sn- glycero-3-phosphocholine (PC-II). Rabbit liver cytochrome b5 was inserted into these vesicles spontaneously and the resulting vesicles containing the cytochrome b5 in the transferable form were photolyzed. Cytochrome b5 containing covalently cross-linked phospholipids was isolated by Sephadex LH-60 column chromatography using ethanol/formic acid as the solvent. Of the total radioactivity, 4.6% (PC-I) or 11.3% (PC-II) was linked to the protein; of the former, up to 51% was base-labile, while in the latter, 22% was base-labile. The sites of cross-linking of PC-I to the protein were investigated by fragmentation with trypsin, Staphylococcus aureas V8 protease, CNBr, and o-iodosobenzoic acid followed by Sephadex LH-60 chromatography and Edman sequencing (solid phase) of the appropriate fragments. The distribution of cross-linking was broad (Ser-104 to Met-130), showing a bell-shaped pattern with a significant peak at Ser-118. The labeling pattern is consistent with the previously proposed loop-back model for the membranous segment in the transferable form of cytochrome b5.

Animals↗
PubMed1983

Long-term marrow culture reveals chromosomally normal hematopoietic progenitor cells in patients with Philadelphia chromosome-positive chronic myelogenous leukemia.

We found that when marrow cells from four patients with newly diagnosed Philadelphia chromosome-positive chronic myelogenous leukemia were maintained in culture for two to four weeks, a previously undetectable population of chromosomally normal hematopoietic cells (including erythroid, granulopoietic, and pluripotent progenitors) became readily demonstrable in three cases. Time-course studies showed that in such cultures the dominant Philadelphia chromosome-positive population rapidly disappeared, in contrast to coexisting chromosomally normal progenitors, which remained detectable for periods of two to three months. Long-term marrow cultures thus offer a new approach to the assessment of a suppressed but functionally intact population of chromosomally normal hematopoietic stem cells in patients with chronic myelogenous leukemia.

Adult↗
PubMed1982

Metastable phase behavior of a sphingolipid analogue.

The phase behavior of the sphingolipid analogue 1-palmitoyl-2-tridecanylcarbamyloxy-sn-glycero-3-phosphocholine (CM-PC) has been studied by differential scanning calorimetry. When CM-PC is cooled at rates greater than 5 K/min, subsequent heating runs exhibit metastable behavior: a low enthalpy exotherm is observed at about 9 degrees C (delta H = -(1-2)kcal/mol), followed by a high enthalpy endotherm at 38 degrees C (delta H = 13 kcal/mol). Systematic variation of cooling/heating protocols indicates that CM-PC exhibits two low temperature states, one metastable and the other stable. Cooling from the liquid crystalline state results in formation of the metastable low-temperature polymorph I, which must transform into the stable low-temperature polymorph II before the liquid crystalline state can be reached again. This metastable thermal behavior is virtually identical to that recently reported for synthetic palmitoyl cerebroside (Ruocco, M.J., Atkinson, D., Small, D.M., Skarjune, R.P., Oldfield, E. and Shipley, G.G. (1981) Biochemistry 20, 5957-5966) and for bovine brain n-acylcerebrosides (Curatolo, W. (1982) Biochemistry, 21, 1761-1764). The observation of the metastable phase behavior of CM-PC indicates that the sphingosine backbone is not a prerequisite for such metastable behavior. Furthermore, the carbamyl group in CM-PC is reversed in orientation compared with the amide of sphingolipids (-NH-CO- vs. -CO-NH-), suggesting that the intermolecular hydrogen bonding potential, rather than some highly specific steric or conformational constraint, is responsible for the observed metastability of sphingolipids.

Calorimetry↗
PubMed1982

The site of attachment of retinal in bacteriorhodopsin. The epsilon-amino group in Lys-41 is not required for proton translocation.

Chymotryptic fragments C-1 (amino acids 72-248) and C-2 (amino acids 1-71) of bacteriorhodopsin have been shown previously to reassociate so as to regenerate the native bacteriorhodopsin chromophore in lipid/detergent mixtures and to form functional proton-translocating vesicles. The fragment C-2 has now been selectively methylated with formaldehyde and sodium cyanoborohydride to give the epsilon-dimethylamino derivatives of Lys-30, 40, and 41 in 96-99% average yield. The methylated and unmethylated C-2 fragments were identical in their ability to reassociate with fragment C-1 and retinal to regenerate the bacteriorhodopsin chromophore and to form functional proton-translocating vesicles. In contrast, dimethylation of the lysine residues of the C-1 fragment gave a derivative which did not form an active complex with unmethylated C-2. We conclude that the epsilon-amino group in Lys-41 is not required for Schiff's base formation with retinal at any step in the light-driven proton-translocation cycle.

Bacteriorhodopsins↗
PubMed1981

Modification of phospholipid structure results in greater stability if liposomes in serum.

Previous studies have revealed that the replacement of the C-2 ester group in phosphatidylcholine by the carbamyloxy function renders the resulting lipids, without affecting the properties of the liposomes, resistant to hydrolysis by phospholipase A2 (Gupta, C.M. Bali, A. (1981) Biochim. Biophys, Acta 663, 506-515). As an extension of this work, the effect of serum on the stability of liposomes, prepared from 1-palmitoyl-2-heptadec-10-cis-enylcarbamyloxyphosphatidylcholine (carbamylphosphatidylcholine), has been examined. The stability has been measured in terms of (a) bilayer permeability to solutes, and (b) the lipid transfer to serum proteins. Replacement of egg phosphatidylcholine in liposomes by the carbamyl analog prevented serum-induced leakage of the entrapped solutes and also inhibited the lipid (phospholipid and cholesterol) transfer. Manipulation of the cholesterol content of the liposomes had no effect on the stability. These observations indicate that the interaction of serum proteins with liposomes probably involves a highly specific binding of the proteins to the liposome surface.

Animals↗
PubMed1981

Transbilayer phospholipid asymmetry in Plasmodium knowlesi-infected host cell membrane.

The membranes from normal and Plasmodium knowlesi-infected rhesus monkey erythrocytes (90 to 95 percent infected with early ring stage) were analyzed for transbilayer distribution of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), by means of chemical and enzymatic probes. The external monolayer of the normal red cell membrane contained at least 68 to 72 percent of the total phosphatidylcholine and 15 to 20 percent of the total phosphatidylethanolamine. In the infected cell, the transmembrane phosphatidylcholine distribution appeared to be reversed, with only 20 to 30 percent of it being externally localized, whereas roughly equal amounts of phosphatidylethanolamine were present in the outer and inner surfaces. However, total phosphatidylethanolamine were present in the outer and inner surfaces. However, total phosphatidylserine in both the infected and normal red cells was exclusively internal. Unlike that in the normal intact cell, external phosphatidylethanolamine in the parasitized cell was readily accessible to phospholipase A2. These results indicate that significant changes in molecular architecture of the host cell membrane are the result of parasitization.

Animals↗