PubMed Health⌕ Search

Biomedical subjects

C F Schmidt

Publications and source records attributed to C F Schmidt.

At least 19 recordsLinked to original sources
PubMed1992

Conformation and elasticity of the isolated red blood cell membrane skeleton.

We studied the structure and elasticity of membrane skeletons from human red blood cells (RBCs) during and after extraction of RBC ghosts with nonionic detergent. Optical tweezers were used to suspend individual cells inside a flow chamber, away from all surfaces; this procedure allowed complete exchange of medium while the low-contrast protein network of the skeleton was observed by high resolution, video-enhanced differential interference-contrast (DIC) microscopy. Immediately following extraction in a 5 mM salt buffer, skeletons assumed expanded, nearly spherical shapes that were uncorrelated with the shapes of their parent RBCs. Judging by the extent of thermal undulations and by their deformability in small flow fields, the bending rigidity of skeletons was markedly lower than that of either RBCs or ghosts. No further changes were apparent in skeletons maintained in this buffer for up to 40 min at low temperatures (T less than 10 degrees C), but skeletons shrank when the ionic strength of the buffer was increased. When the salt concentration was raised to 1.5 M, shrinkage remained reversible for approximately 1 min but thereafter became irreversible. When maintained in 1.5 M salt buffer for longer periods, skeletons continued to shrink, lost flexibility, and assumed irregular shapes: this rigidification was irreversible. At this stage, skeletons closely resembled those isolated in standard bulk preparations. We propose that the transformation to the rigid, irreversibly shrunken state is a consequence of spectrin dimer-dimer reconnections and that these structural rearrangements are thermally activated. We also measured the salt-dependent size of fresh and bulk extracted skeletons. Our measurements suggest that, in situ, the spectrin tethers are flexible, with a persistence length of approximately 10 nm at 150 mM salt.

Elasticity↗
PubMed1992

Analysis of cholesterol in all lipoprotein classes by single vertical ultracentrifugation of fingerstick blood and controlled-dispersion flow analysis.

This new, highly sensitive analytical system, based on controlled dispersion of the flowing sample, gives a rapid, continuous, and direct analysis for cholesterol in all lipoprotein classes, separated by single vertical-spin density-gradient ultracentrifugation. In this Vertical Auto Profile-II fingerstick system, designated VAP-IIfs, a narrow-bore Teflon coil serves as the reactor with no segmentation of the analytical stream by air bubbles, in contrast to the Technicon AutoAnalyzer used in the VAP-I method. Concentrations of high-, low-, intermediate-, and very-low-density lipoprotein cholesterol and lipoprotein(a) cholesterol are determined by decomposing the spectrophotometric absorbance curve for the continuous analysis of the centrifuged sample, with use of software developed in this laboratory. Total cholesterol is determined from the total area under the absorbance curve. For assaying total cholesterol, the CV between aliquots within a rotor ranged from 1.35% to 3.15%; the CV between rotors was 2.45%. Because only 18 microL of sample is required, VAP-IIfs can be readily adapted to analysis for lipoprotein cholesterol profiles in capillary blood samples. Total cholesterol values by VAP-IIfs for fingerstick and venous samples from 23 subjects agreed well: slope = 1.01 (SD 0.03), intercept = -21 (SD 51) mg/L, Sy/x = 50 mg/L, and r = 0.992. Results by VAP-IIfs also correlated highly with results for duplicate samples analyzed at the Northwest Lipid Research Laboratories.

Cholesterol↗
PubMed1990

Multilayer adsorption of lysozyme on a hydrophobic substrate.

Macromolecular adsorption is known to occur as a complex process, often in a series of steps. Several models are discussed in the literature which describe the microscopic structure of the adsorbate. In the present study we investigated the adsorption of hen egg white lysozyme on alkylated silicon oxide surfaces. A combination of fluorescence excitation in the evanescent field and fluorescence recovery after photobleaching allowed us to measure the amount of adsorbed fluorescent lysozyme and the equilibrium exchange kinetics with molecules in solution. We found that a model with at least three classes of adsorbed molecules is necessary to describe the experimental results. A first layer is formed by the molecules which adsorb within a short time after the beginning of the incubation. These molecules make up approximately 65% of the final coverage. They are quasi-irreversibly adsorbed and do not measurably exchange with bulk molecules within one day even at temperatures up to 55 degrees C. A second layer, which reaches equilibrium only after several hours of incubation, shows a pronounced exchange with bulk molecules. The on-off kinetics show a distinct temperature dependence from which an activation barrier of delta E approximately 22 kcal/mol is derived. A third layer of molecules that exchange rapidly with the bulk can be seen to comprise approximately 10% of the total coverage. The exchange rate is on the order of fractions of a second. The binding of the latter two classes of adsorbed molecules is exothermic. From the temperature dependence of the coverage, the binding enthalpy of the slowly exchanging layer was estimated to be delta Hads approximately 3.8 kcal/mol. The second and third class of molecules remain enzymatically active as a muramidase, which was tested by the lysis of the cell walls of Micrococcus lysodeiktikus. The molecules in the first layer, on the other hand, showed no enzymatic activity.

4-Chloro-7-nitrobenzofurazan↗
PubMed1985

Studies of synthetic peptide analogs of the amphipathic helix. Structure of complexes with dimyristoyl phosphatidylcholine.

The amphipathic helix hypothesis for the lipid-associating domains of exchangeable plasma apolipoproteins has been further studied by analysis of the structure of the complexes formed between four synthetic peptide analogs of the amphipathic helix and dimyristoyl phosphatidylcholine (DMPC). Density gradient ultracentrifugation, negative stain electron microscopy, nondenaturing gradient gel electrophoresis, 1H NMR, high sensitivity differential scanning calorimetry, and circular dichroism were the techniques used in these studies. The two analogs Asp-Trp-Leu-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Leu-Lys-Glu-Ala-Phe (18A) and 18A-Pro-18A whose sequences most strongly mimic native amphipathic sequences were found also most strongly to mimic apolipoprotein A-I in DMPC complex structure. The covalently linked dimer of the prototype amphipathic analog 18A, 18A-Pro-18A, appears to have greater lipid affinity than 18A. This presumably is the result of the cooperativity provided by two covalently linked lipid-associating domains in 18A-Pro-18A. The studies further suggest that the charge-reversed analog of the prototype 18A, reverse-18A, has the lowest lipid affinity of the four analogs studied and forms only marginally stable discoidal DMPC complexes. We postulate that this low lipid affinity is due predominantly, but not necessarily exclusively, to the lack of a hydrophobic contribution of lysine residues at the polar-nonpolar interface of reverse-18A versus 18A. The intermediate lipid affinity of des-Val10-18A, the fourth analog peptide, to produce a rank order of 18A-Pro-18A greater than 18A greater than des-Val10-18A greater than reverse-18A, supports this interpretation. Des-Val10-18A which has Val deleted from 18A has an amphipathic helical structure partially disrupted by the shift of 2 lysine residues away from the polar-nonpolar interface.

Amino Acid Sequence↗
PubMed1981

Vesicle- vesicle interactions in sonicated dispersions of dipalmitoylphosphatidylcholine.

The time course of the size transformation of sonicated small unilamellar dipalmitoylphosphatidylcholine vesicles at 23 degrees C has been followed with (31)P and (1)H nuclear magnetic resonance (NMR) spectroscopy. Comparison of these results with turbidity measurements indicates that vesicle aggregation, monitored by turbidity, and size transformation, followed by NMR, occur on distinctly different time scales. For lipid concentrations in the 5-50 mM range, aggregation takes place on a time scale of minutes, whereas size transformation takes many hours. Aggregation, unlike size transformation, can be reversed by increasing the temperature above the phospholipid phase transition temperature. Analysis of the (31)P NMR line shapes provides evidence for a model in which the small vesicles transform into the product vesicles [characterized by Schullery, S.E., Schmidt, C.F., Felgner, P., Tillack, T.W., & Thompson, T.E. (1980) Biochemistry 19, 3919] without producing significant amounts of intermediate-size vesicles. Kinetic analysis indicates that the size transformation is apparently second order. (1)H NMR data indicate that the rate of transformation is decreased if trivalent ions are added to the dispersions and also if the temperature is periodically increased above the transition temperature. Analysis of the latter experiment provides some evidence that vesicle aggregation is a necessary precursor to size transformation. It was also found that increasing the average vesicle size decreases the extent of transformation.

Kinetics↗
PubMed1981

Solid-state carbon-13 nuclear magnetic resonance of the lecithin gel to liquid-crystalline phase transition.

The temperature dependence of the 13C NMR spectra of dipalmitoylphosphatidylcholine (DPPC) which has been 13C labeled at the carbonyl position of the sn-2 chain, 2-[1-13C]DPPC, is reported. In the L beta' phase an axially symmetric spectrum of 112-ppm breadth is observed, and this transforms to an isotropic-like line ((delta sigma) approximately 7 ppm) in the L alpha phase. In the intermediate P beta phase a temperature-dependent superposition of these spectra is observed, which suggests that this phase exhibits microscopic structural and dynamical properties of both the L beta' and L alpha phases. An analysis of the spectral line shapes leads to the conclusion that the appearance of the isotropic-like line in the P beta' phase is primarily due to a conformational change at the sn-2 carbonyl which is complete at the main transition. Increased rates of axial diffusion in the P beta' phase may contribute to the narrowing.

Carbon Isotopes↗
PubMed1981

Effect of surface curvature on stability, thermodynamic behavior, and osmotic activity of dipalmitoylphosphatidylcholine single lamellar vesicles.

The size and surface curvature dependence of the properties and stability of single lamellar vesicles have been investigated by using a variety of physicochemical techniques. Dipalmitoylphosphatidylcholine single lamellar vesicles of sizes ranging between 200 and 900 A in diameter have been prepared by the French press method and characterized with respect to their size distribution, stability, and thermotropic behavior by negative stain electron microscopy, molecular sieve chromatography, nuclear magnetic resonance spectroscopy, and differential scanning calorimetry. Vesicles with a diameter smaller than 400 A are unstable below their transition temperature and fuse spontaneously to form larger single lamellar vesicles. Correlation analysis of experimentally obtained size distributions and calorimetric phase transitions profiles allowed estimation of the size dependence of the transition temperature. The phase transition temperature depends on the vesicle size in a sigmoidal fashion. Throughout the entire 200-700 A diamter range, the phase transition parameters are sensitive to size; however, the size dependence is especially pronounced around 400 A in diameter. The anomalous size dependence of the transition temperature for vesicles smaller than 400 A in diameter has been attributed to a decrease in the effective bilayer curvature due to packing rearrangements of the lipid molecules. Changes in the fractional degree of self-quenching of trapped 6-carboxyfluorescein induced by osmotic stress indicate that large single lamellar vesicles are not spherical under isoosmotic conditions. These vesicles are relatively flexible and can sustain almost a 2-fold increase in their internal aqueous volume without any leakage of the internal content.

Drug Stability↗
PubMed1980

Fusion of dipalmitoylphosphatidylcholine vesicles.

Small unilamellar dipalmitoylphosphatidylcholine vesicles formed by sonication are shown to fuse spontaneously below the phase transition temperature. The ultimate fusion products are unilamellar vesicles about 700 A in diameter, which are stable and provide an intact ionic permeation barrier either above or below the phase transition. The fused vesicles have been characterized by gel chromatography, trapped volume, 31P nuclear magnetic resonance, and negative stain and freeze-fracture electron microscopy.

Chromatography, Gel↗
PubMed1977

A nuclear magnetic resonance study of sphingomyelin in bilayer systems.

The physical properties of small single-walled vesicles composed of the zwitterionic phospholipid sphingomyelin have been studied using 1H and 31P nuclear magnetic resonance spectroscopy. The temperature variation of proton line widths and spin-lattice relaxation times and the chemical shift behavior for sphingomyelin vesicles are compared with results previously determined for phosphatidylcholine vesicles. Differences between the two systems are interpreted as indications of the presence of both inter- and intramolecular hydrogen bonding in sphingomyelin bilayers.

Animals↗
PubMed1975

Utilization of an Escherichia coli mutant for carbon-13 enrichment of tRNA for NMR studies.

The enrichment of tRNA at specific sites with carbon-13 has been accomplished in vivo using a mutant of Escherichia coli. A relaxed strain of E. coli auxotrophic for methionine was grown in a specifically defined medium supplemented with either [14C] or [13C]-methyl labeled methionine. Cells were collected at the end of the log-phase of growth and tRNA was extracted. Analysis of the radioactivity of the [14C]-labeled tRNA established an incorporation ratio of three labeled carbons per tRNA molecule. Incorporation of the [14C]-label in vivo was confined to the methylation of nucleotides as determined by thin layer chromatography of nucleotides resulting from a ribonuclease digestion of [14C]-labeled tRNA. The carbon-13 NMR spectrum of [13C]-enriched tRNA indicated a similar degree of incorporation into the methylated nucleotides by the substantial enhancement of [13C]-methyl NMR signals only. Assignment of signals has been made for the methyl groups of ribothymidine and N7-methylguanosine in E. coli tRNA.

Carbon Isotopes↗
PubMed1971

Enrichment, isolation, and cultural characteristics of marine strains of Clostridium botulinum type C.

Terrestrial strains of Clostridium botulinum type C, designated 468 and 571, were used to screen various media for growth and sporulation at 30 C. Of the various formulations tested, only egg meat medium fortified with 1% additions of yeast extract, ammonium sulfate, and glucose (FEM medium) gave good growth and satisfactory sporulation. FEM medium was used to recover four marine type C isolates from inshore sediments collected along the Atlantic, the Gulf of Mexico, and the Pacific coasts of the United States. The isolation techniques involved repeated transfer of cultures showing type C toxin in FEM medium and purification by a deep tube method. The medium used for purification was beef infusion-agar supplemented with 0.14% sodium bicarbonate and 0.1% l-cysteine hydrochloride. l-Cysteine was adopted in preference to sodium thioglycolate, because some lots of the latter were definitely inhibitory for growth. The addition of bicarbonate markedly increased viable spore counts of both the marine and terrestrial strains. Various cultural and biochemical characteristics of the marine and the terrestrial strains were compared. With the exception of some variations in their fermentation patterns, both groups showed similar characteristics. Of 23 fermentable compounds tested, the terrestrial strains attacked only glucose and mannose. The marine strains fermented glucose, mannose, galactose, and ribose actively; dextrin, inositol, maltose, and melibiose were weakly fermented.

Ammonium Sulfate↗
PubMed1971

Minimal growth temperature, sodium chloride tolerance, pH sensitivity, and toxin production of marine and terrestrial strains of Clostridium botulinum type C.

Minimal growth temperatures of four marine and two terrestrial strains of Clostridium botulinum type C were determined in a laboratory culture medium, fortified egg meat medium (FEM), and in ground haddock. The inoculum equaled 2 x 10(6) viable spores per tube with five-tube replicate sets. The spores were preheated in aqueous suspension at 71 C for 15 min prior to inoculation to reduce toxin carry-over. Similar results were obtained in both substrates. Both the marine and the terrestrial strains grew at 15.6 C, but only the terrestrial strains grew at 12.8 C. None of the strains grew at 10 C during prolonged incubation. The sodium chloride tolerance and the pH sensitivity of the marine and the terrestrial strains were determined at 30 C. The basal medium consisted of beef infusion broth. The inoculum level equaled 2 x 10(6) unheated spores per replicate. Growth was inhibited at salt concentrations from 2.5 to 3.0%. The terrestrial strains were more pH-sensitive than the marine strains. Whereas the terrestrial strains failed to grow below pH 5.62, three of the marine strains grew at pH 5.10, but not at pH 4.96, during extended incubation. One marine strain grew at pH 5.25, but not below. FEM and proteose peptone-Trypticase-yeast extract-glucose medium permitted the production of high levels of botulinum toxin among four media tested. Toxin produced by the marine and terrestrial strains showed no increase in toxicity after incubation with trypsin.

Agar↗