Biomedical subjects

J D Baldeschwieler

Publications and source records attributed to J D Baldeschwieler.

At least 19 recordsLinked to original sources

PubMed1996

Evidence for the role of proteoglycans in cation-mediated gene transfer.

We report evidence that gene complexes, consisting of polycations and plasmid DNA enter cells via binding to membrane-associated proteoglycans. Treatment of HeLa cells with sodium chlorate, a potent inhibitor of proteoglycan sulfation, reduced luciferase expression by 69%. Cellular treatment with heparinase and chondroitinase ABC inhibited expression by 78% and 20% with respect to control cells. Transfection was dramatically inhibited by heparin and heparan sulfate and to a smaller extent by chondroitan sulfate B. Transfection of mutant, proteoglycan deficient Chinese hamster ovary cells was 53 x lower than of wild-type cells. For each of these assays, the intracellular uptake of DNA at 37 degrees C and the binding of DNA to the cell membrane at 4 degrees C was impaired. Preliminary transfection experiments conducted in mutant and wild-type Chinese hamster ovary cells suggest that transfection by some cationic lipids is also proteoglycan dependent. The variable distribution of proteoglycans among tissues may explain why some cell types are more susceptible to transfection than others.

Animals↗

PubMed1996

Receptor-mediated endocytosis of poly(acrylic acid)-conjugated liposomes by macrophages.

The uptake characteristics of negatively-charged liposomes made by conjugation of poly(acrylic acid) (PAA) were studied with respect to cultured RAW macrophages. The PAA-conjugated liposomes were internalized and digested in an acidic compartment at a much faster rate than the unmodified phosphatidylcholine (PC) liposomes. After incubation for 18 h, an over 5-fold increase in the uptake of PC liposomes was obtained by PAA conjugation. Subsequently, part of the aqueous phase of the internalized liposomes was exocytosed. Recognition of PAA by the macrophages seems to be responsible for the enhanced uptake of PAA-conjugated liposomes. Cross-competition experiments showed that PAA-conjugated liposomes inhibited the uptake of acetylated-low density lipoprotein (acetyl-LDL) by the macrophages and vice versa. The uptake of PAA-conjugated liposomes was also inhibited by dextran sulfate and maleylated-bovine serum albumin (maleyl-BSA), which are also known to bind to scavenger receptors. Poly(C) and BSA, which are not ligands for the scavenger receptor, competed poorly with the uptake of PAA-conjugated liposomes. Enhanced uptake of PAA-conjugated liposomes by CHO cells with low scavenger receptor expression was not observed. Unexpectedly, LDL, which is not a ligand for scavenger receptor, also partially inhibited the uptake of PAA-conjugated liposomes. The interaction of PAA-conjugated liposomes with macrophages is complex, and the endocytosis of PAA-conjugated liposomes most likely involves multiple receptors and/or pathways. The data obtained suggest that the high affinity binding of PAA-conjugated liposomes to macrophages may be due to recognition of the negative charges of PAA by cell surface receptors, including the scavenger receptor.

Acrylic Resins↗

PubMed1995

Real-time detection of DNA hybridization and melting on oligonucleotide arrays by using optical wave guides.

The challenge of the Human Genome Project is to increase the rate of DNA sequence acquisition by two orders of magnitude to complete sequencing of the human genome by the year 2000. The present work describes a rapid detection method using a two-dimensional optical wave guide that allows measurement of real-time binding or melting of a light-scattering label on a DNA array. A particulate label on the target DNA acts as a light-scattering source when illuminated by the evanescent wave of the wave guide and only the label bound to the surface generates a signal. Imaging/visual examination of the scattered light permits interrogation of the entire array simultaneously. Hybridization specificity is equivalent to that obtained with a conventional system using autoradiography. Wave guide melting curves are consistent with those obtained in the liquid phase and single-base discrimination is facile. Dilution experiments showed an apparent lower limit of detection at 0.4 nM oligonucleotide. This performance is comparable to the best currently known fluorescence-based systems. In addition, wave guide detection allows manipulation of hybridization stringency during detection and thereby reduces DNA chip complexity. It is anticipated that this methodology will provide a powerful tool for diagnostic applications that require rapid cost-effective detection of variations from known sequences.

Base Sequence↗

PubMed1995

Atomic force microscopy of human hair cuticles: a microscopic study of environmental effects on hair morphology.

We have used an atomic force microscope to provide quantitative real-time analysis of human hair morphologic changes under ambient conditions. This form of microscopy combines the lateral resolution of an electron microscope and the flexibility of a light microscope. Three experiments were performed: a study of hair morphology in air versus water, a kinetic study of hair hydration, and a determination of how pH changes affect hair morphology. The overlapping keratinized cells that form the hair cuticle spread out between 50 and 150% when hydrated, compared to a total shaft diameter change of 10%. This hydration reaches a saturation point within the first few minutes after immersion. Also, hair swells much more at higher pH.

Adult↗

PubMed1993

Organ distributions of liposome-loaded rat platelets.

Past in vitro functional assays have demonstrated that platelet function is not inhibited by liposome uptake. In the present study, the organ distributions of control and liposome-loaded Sprague-Dawley rat platelets were examined to determine whether liposome uptake enhances RES uptake. Platelets were isolated using STRactan density gradient centrifugation, incubated with small unilamellar liposomes in vitro for 1 hour, labeled with 51Cr and injected into a cohort group of rats. One hour post-injection the spleen, liver, lungs, blood, kidneys and bladder contents were removed and the percentages of the recovered dose localized per total organ (%RD) were determined. The RES index, defined as %RDliver + %RDspleen, were 24.8 +/- 4.5 and 20.5 +/- 5.0 for the control platelets and liposome-loaded platelets, respectively. These results indicate that liposome uptake does not enhance RES uptake.

Animals↗

PubMed1993

Subfibrillar structure of type I collagen observed by atomic force microscopy.

We have imaged native rat tail and reconstituted bovine dermal type I collagen by atomic force microscopy, obtaining a level of detail comparable to that obtained on the same samples by transmission electron microscopy. The characteristic 60-70 nm D periodicity consists of ridges exhibiting high tip-sample adhesion alternating with 5-15-nm-deep grooves having low adhesion. We also observe an intraperiod or "minor" band consisting of 1-nm-deep grooves, and "microfibrils" arranged parallel to or inclined approximately 5 degrees to the fibril axis. In air collagen fibrils exhibit negligible compression under the forces exerted by the tip. When immersed in water the subfibrillar features disappear and the fibrils become softer, compressing by 5% of their height under an 11-nN force. Material on the surface of the sample sometimes accumulates on the atomic force microscope tip; contrary to expectation such tip contamination can improve as well as reduce resolution.

Animals↗

PubMed1992

Phagocytosis of liposomes by human platelets.

We have shown that platelets are capable of phagocytosing liposomes rather than simply sequestering particles as previously postulated. Incubation of human platelets with small neutral unilamellar liposomes (approximately 74 nm) resulted in uptake of the liposomes and retention of the lipid with rapid release of the aqueous-phase components. The lipid label [3H]-cholesterylhexadecyl ether and water-soluble [3H]inulin were used to study the fate of the liposome components. Uptake of liposomes was proportional to the number of liposomes added and to the incubation time. Approximately 250 liposomes per platelet were taken up within a 5-hr incubation period. Uptake of the liposomes occurred through the open-channel system, as evidenced by thin-section electron microscopy, and was followed by accumulation and degradation in acid- and esterase-containing vesicles, as determined by changes in fluorescence of the pH-sensitive probe, pyranine (1-hydroxypyrene-3,6,8-trisulfonic acid), and hydrolysis of the cholesteryl [14C]oleate membrane marker. Uptake was inhibited by the addition of EDTA, cytochalasin B, or 2,4-dinitrophenol and iodoacetate to the medium. Results from the serotonin release assay, micro-aggregation assay, fluorescein diacetate membrane integrity assay, and electron microscopy indicate that neither the conditions for loading nor phagocytosis of liposomes significantly alter platelet function or morphology.

Blood Platelets↗

PubMed1992

Membrane fusion of mumps virus with ghost erythrocytes and CV-1 cells.

The octadecyl rhodamine (R18) fluorescent dequenching assay was used to examine membrane fusion between mumps virus and mammalian cells. Rapid fluorescent dequenching, indicative of membrane fusion, was observed when labeled mumps virus was mixed with either ghost erythrocytes or CV-1 cells. After 15 min a saturation limit of 18 virus per erythrocyte ghost and 6400 virus per CV-1 cell was observed. Fetuin was found to inhibit virus fusion, suggesting a role for sialic acid in virus binding to the cells. Two dequenching processes were observed of which the faster process is thought to be membrane fusion and the second process is thought to be probe proximal transfer.

Cell Line↗

PubMed1991

Alterations in membrane surfaces induced by attachment of carbohydrates.

We have examined the behavior of the dry phospholipid dipalmitoylphosphatidylcholine (DPPC) in the presence of several carbohydrate derivatives. These carbohydrate derivatives possess a hydrophobic portion which is incorporated directly into the DPPC membrane and a hydrophilic portion which places the carbohydrate structure at the membrane interface with the surrounding matrix. In the presence of these derivatives, the physical properties of the membrane are altered. These alterations are evident in changes observed in the phosphate and carbonyl vibrational modes of the phospholipid portion of the membrane. In addition, the phase transition behavior of the lipid is significantly altered as evidenced by a reduction in the gel to liquid-crystalline phase transition temperature. These results are consistent with those previously reported for free carbohydrates interacting with membranes in which a water replacement hypothesis has been used to explain the behavior. The attachment of carbohydrates to the membrane enhances these effects by localizing the agent responsible for these alterations at the membrane interface.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1991

The cryoprotective action of synthetic glycolipids.

Egg PC vesicles frozen and thawed in the presence of carbohydrate derivatives do not incur damage normally associated with freeze thawing. Treated vesicles maintain membrane integrity as evidenced by the lack of lipid intermixing and maintenance of vesicle size following freezing and thawing. This protection is conferred at a derivative:lipid ratio of 0.4 mol/mol, significantly lower than the amount of carbohydrate required when not attached directly to the vesicle. This result indicates that only the carbohydrate at the vesicle surface is responsible for imparting stability to the membrane. This effect can be modulated by variations in the nature of the surrounding medium or alterations in the structure of the carbohydrate, suggesting that direct interactions between the carbohydrate and membrane occur at the membrane interface which are sensitive to bulk phase properties.

Cryoprotective Agents↗

PubMed1990

Atomic-scale imaging of DNA using scanning tunnelling microscopy.

The scanning tunnelling microscope (STM) has been used to visualize DNA under water, under oil and in air. Images of single-stranded DNA have shown that submolecular resolution is possible. Here we describe atomic-resolution imaging of duplex DNA. Topographic STM images of uncoated duplex DNA on a graphite substrate obtained in ultra-high vacuum are presented that show double-helical structure, base pairs, and atomic-scale substructure. Experimental STM profiles show excellent correlation with atomic contours of the van der Waals surface of A-form DNA derived from X-ray crystallography. A comparison of variations in the barrier to quantum mechanical tunnelling (barrier-height) with atomic-scale topography shows correlation over the phosphate-sugar backbone but anticorrelation over the base pairs. This relationship may be due to the different chemical characteristics of parts of the molecule. Further investigation of this phenomenon should lead to a better understanding of the physics of imaging adsorbates with the STM and may prove useful in sequencing DNA. The improved resolution compared with previously published STM images of DNA may be attributable to ultra-high vacuum, high data-pixel density, slow scan rate, a fortuitously clean and sharp tip and/or a relatively dilute and extremely clean sample solution. This work demonstrates the potential of the STM for characterization of large biomolecular structures, but additional development will be required to make such high resolution imaging of DNA and other large molecules routine.

Animals↗

PubMed1989

Effect of cholesterol on the uptake and intracellular degradation of liposomes by liver and spleen; a combined biochemical and gamma-ray perturbed angular correlation study.

We investigated the effect of cholesterol on the uptake and intracellular degradation of liposomes by rat liver and spleen macrophages. Multilamellar vesicles (MLV) consisting of distearoylphosphatidylcholine/phosphatidylserine (molar ratio 9:1) or distearoylphosphatidylcholine/cholesterol/phosphatidylserine (molar ratio 4:5:1) were labeled with [3H]cholesteryl hexadecyl ether and/or cholesteryl [14C]oleate. After i.v. injection the cholesterol-containing liposomes were eliminated less rapidly from the bloodstream and taken up to a lesser extent by the liver (macrophages) than the cholesterol-free liposomes. Assessment of the 3H/14C ratios in liver and spleen cells revealed that the cholesterol-containing liposomes are substantially more resistant towards intracellular degradation than the cholesterol-free liposomes. These results could be confirmed by measuring the release of 111In from liposomes after uptake by liver and spleen by means of gamma-ray perturbed angular correlation spectroscopy. Experiments with cultured Kupffer cells in monolayer also revealed that incorporation of cholesterol results in a decrease of the uptake and an increase of the intracellular stability of cholesteryl [14C]oleate-labeled liposomes. Finally, incubation of both types of liposomes with lysosomal fractions prepared from rat liver demonstrated a difference in susceptibility to lysosomal degradation: the cholesterol-free vesicles were much more sensitive to lysosomal esterase than the cholesterol-containing liposomes. These results may be relevant to the application of liposomes as a drug carrier system to liver and spleen (macrophages).

Animals↗

PubMed1988

Fluorescence measurement of the kinetics of DNA injection by bacteriophage lambda into liposomes.

Bacteriophage lambda attaches to Gram-negative bacteria using the outer membrane protein LamB as its receptor. Subsequently, DNA is injected by the bacteriophage into the host cell for replication and expression. The mechanism of DNA injection, however, is poorly understood. In order to begin to characterize DNA injection, a quantitative kinetic assay to detect injection into reconstituted LamB liposomes is described. The technique involves monitoring the increase in fluorescence of liposome-encapsulated ethidium bromide, which occurs as DNA enters the aqueous compartment of the vesicles. The data indicate that injection is several times faster than indicated by earlier studies and is complete within 1 min. Such assays which allow direct observation of this process are necessary first steps toward a mechanistic understanding.

Bacterial Outer Membrane Proteins↗

PubMed1988

Modification of lipid phase behavior with membrane-bound cryoprotectants.

Several derivatives of cholesterol containing oxyethylene headgroups with and without a terminal galactose have been synthesized in order to examine the effects of immobilizing a cryoprotectant at a membrane surface. In this work, we have studied the ability of the triethoxycholesterol (TEC) and triethoxycholesterol galactose (TEC-Gal) derivatives to modulate the phase behavior of phosphatidylcholine and phosphatidylethanolamine membranes. Methods of fluorescence polarization, 31P-NMR and freeze-fracture electron microscopy were employed to monitor these changes in lipid phase behavior. Fluorescence polarization data demonstrated the ability of the derivatives to fluidize gel state and rigidify liquid-crystalline state phosphatidylcholines in a manner similar to that observed for cholesterol. Unlike cholesterol, however, the Tm of dipalmitoylphosphatidylcholine (DPPC) was reduced in a concentration-dependent manner with each of the derivatives. Freeze-fracture electron microscopy and 31P-NMR of DOPE dispersions indicate an increase in the lamellar to hexagonal phase-transition temperature on the order of 10-20 C degrees above room temperature for mixtures with 20 mol% of the derivatives. These results are discussed in terms of the properties exhibited by compounds such as carbohydrates, which are known to serve as cryoprotectants for synthetic and biological membranes.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1988

In vivo stability of ester- and ether-linked phospholipid-containing liposomes as measured by perturbed angular correlation spectroscopy.

To evaluate liposome formulations for use as intracellular sustained-release drug depots, we have compared the uptake and degradation in rat liver and spleen of liposomes of various compositions, containing as their bulk phospholipid an ether-linked phospholipid or one of several ester-linked phospholipids, by perturbed angular correlation spectroscopy. Multilamellar and small unilamellar vesicles (MLVs and SUVs), composed of egg phosphatidylcholine, sphingomyelin, distearoyl phosphatidylcholine (DSPC), dipalmitoyl phosphatidylcholine (DPPC) or its analog dihexadecylglycerophosphorylcholine (DHPC), and cholesterol plus phosphatidylserine, and containing 111In complexed to nitrilotriacetic acid, were injected intravenously in rats. Recovery of 111In-labeled liposomes in blood, liver, and spleen was assessed at specific time points after injection and the percentage of liposomes still intact in liver and spleen was determined by measurement of the time-integrated angular perturbation factor [G22(infinity)] of the 111In label. We found that MLVs but not SUVs, having DHPC as their bulk phospholipid, showed an increased resistance against lysosomal degradation as compared to other phospholipid-containing liposomes. The use of diacyl phospholipids with a high gel/liquid-crystalline phase-transition temperature, such as DPPC and DSPC, also retarded degradation of MLV, but not of SUV in the dose range tested, while the rate of uptake of these liposomes by the liver was lower.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1987

Mouse erythrocyte carriers osmotically loaded with methotrexate.

The mouse red blood cell (RBC) and red blood cell ghost (RBCG) have been studied as carriers of methotrexate (MTX). When incubated with high concentrations of MTX, RBCs take up significant quantities of it. However, when active loading techniques, such as the slow dialysis and preswell methods, are applied to those cells, up to 15 times more MTX can be entrapped. We have studied factors critical to the incorporation, leakage, and morphology of RBCGs during their loading with MTX by the slow dialysis and preswell methods. Compounds added to the buffers to maintain the ATP content of the cells and osmolarity play functional roles in this process. The fate of the material entrapped within the ghosts after in vivo administration was shown to be capture by the reticuloendothelial system. The pharmacological efficacy of MTX-loaded RBCGs in treating mice bearing hepatoma ascites tumors was demonstrated by increases in average survival time of 28.5-42.8%.

Animals↗

PubMed1985

Modification of vesicle surfaces with amphiphilic sterols. Effect on permeability and in vivo tissue distribution.

In this paper, we describe the permeability of vesicles prepared with various synthetic cholesterol derivatives. Cholesterol derivatives with side-chains ending in hydroxyl groups reduced the permeability of unilamellar vesicles. However, addition of cholesterol derivatives with terminal amino groups makes the vesicles more permeable. Vesicles prepared with a short-chain amino-cholesterol derivative were found to be less permeable in phosphate-buffered saline, but not in bovine serum, while long-chain amino-cholesterol-containing vesicles were very permeable in both media. Studies in vivo indicate a rapid clearance rate for intravenously administered amino-cholesterol-containing vesicles with a concomitant increase in liver uptake. However, no difference was found in either the clearance or tissue distribution of control vesicles and the less permeable hydroxyl-cholesterol-containing vesicles.

Animals↗

PubMed1985

Phospholipid vesicle targeting using synthetic glycolipid and other determinants.

The interactions of carbohydrate-modified phospholipid vesicles with various isolated cell types in vitro have been studied to establish a better basis for understanding the mechanisms for recognition and transport of such modified vesicles in vivo. The physical basis for the use of perturbed angular correlation spectroscopy for the measurement of phospholipid vesicle integrity, and the kinetics of uptake of modified phospholipid vesicles by mouse peritoneal macrophage are first reviewed. The effects of variation of the chemical structure of the determinant and other factors indicate that the rate of uptake of cationic vesicles by mouse peritoneal macrophage is directly related to the distance that an amine group can be extended beyond the vesicle surface, and not, for example, to the stereochemistry of the carbohydrate determinant. The uptake mechanism appears to involve generalized phagocytosis and not a receptor-mediated mechanism, or an opsonization process that is not stereospecific.

Amino Sugars↗