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Biomedical subjects

R Mendelsohn

Publications and source records attributed to R Mendelsohn.

At least 19 recordsLinked to original sources
PubMed1992

Sarcoplasmic reticulum from rabbit and winter flounder: temperature-dependence of protein conformation and lipid motion.

A comparative study of lipids and proteins in sarcoplasmic reticulum (SR) from rabbit and flounder has been undertaken. The protein/phospholipid ratio (w/w) was 3:1 in flounder SR (FSR) and 2.2:1 in rabbit SR (RSR). Both membranes had similar contents of PC (70%) and PI (6%). PE constituted 15% in RSR and 21% in FSR. PS and sphingomyelin were minor components of both SR (less than 4%). There were differences in the unsaturated chains of the total lipid extracts, PC, PE, and PI between FSR and RSR. RSR was high in linoleate and arachidonate while FSR contained substantial amounts of eicosapentaenoate and docosahexaenoate. FTIR spectroscopy revealed that the lipids of both membranes did not undergo a phase transition between 0 and 50 degrees C. The lipids were in the liquid-crystalline state at physiological temperatures and underwent monotonic increases in conformational disorder as the temperature was raised. CD spectra indicated higher content of alpha-helical structure of proteins in RSR than in FSR. Increasing temperature caused diminution of alpha-helix content. Relatively large decreases in ellipticity were observed between 20 degrees C and 40 degrees C for FSR and 30 degrees C and 60 degrees C for RSR. Measurements of intrinsic tryptophan fluorescence as a function of temperature gave similar results for membrane proteins in both FSR and RSR. The rate of change of tryptophan fluorescence and fluorescence lifetimes was constant over the temperature ranges studied, and no abrupt shifts in fluorescence occurred in the temperature regions where ellipticity decreased rapidly.

Animals↗
PubMed1992

Fluorescence, CD, attenuated total reflectance (ATR) FTIR, and 13C NMR characterization of the structure and dynamics of synthetic melittin and melittin analogues in lipid environments.

The structure and dynamics of synthetic melittin (MLT) and MLT analogues bound to monomyristoylphosphatidylcholine micelles, dimyristoylphosphatidylcholine vesicles, and diacylphosphatidylcholine films have been investigated by fluorescence, CD, attenuated total reflectance (ATR) FTIR, and 13C NMR spectroscopy. All of these methods provide information about peptide secondary structure and/or about the environment of the single tryptophan side chain in these lipid environments. ATR-FTIR data provide additional information about the orientation of helical peptide segments with respect to the bilayer plane. Steady-state fluorescence anisotropy, fluorescence lifetime, and 13C NMR relaxation data are used in concert to provide quantitative information about the dynamics of a single 13C-labeled tryptophan side chain at position 19 in lipid-bound MLT, and at positions 17, 11, and 9, respectively, in lipid-bound MLT analogues. Peptide chain dynamics are probed by NMR relaxation studies of 13C alpha-labeled glycine incorporated into each of the MLT peptides at position 12. The cumulative structural and dynamic data are consistent with a model wherein the N-terminal alpha-helical segment of these peptides is oriented perpendicular to the bilayer plane. Correlation times for the lysolipid-peptide complexes provide evidence for binding of a single peptide monomer per micelle. A model for the membranolytic action of MLT and MLT-like peptides is proposed.

Circular Dichroism↗
PubMed1992

An FT-IR microscopic investigation of the effects of tissue preservation on bone.

Fourier transform infrared microscopy is a powerful tool for the characterization of mineral and protein in histologic sections of bone. This study was concerned with determining whether techniques used to preserve these tissues and to prepare them for sectioning had an effect on spectral properties. The nu 1, nu 3 phosphate bands in the 900-1200 cm-1 spectral region were used to evaluate the structure of the apatitic mineral in fresh-frozen, ethanol-fixed, and formalin-fixed 35-day-old rat femurs; fresh-frozen and formalin-fixed 20-day-old fetal rat femurs; ground 35-day-old rat diaphyseal bone samples; and formalin-fixed, methacrylate-embedded ground diaphyseal bone. The crystallinity (crystal size and perfection) of the bone apatite was assessed by a curve-fitting analysis of the nu 1, nu 3 phosphate bands. Results indicate that ethanol or formalin fixation of the 35-day-old intact rat femur, and formalin fixation and embedding of the ground rat bone do not significantly alter the crystallinity of the apatite. However, formalin fixation of the fetal rat bone did alter the structure of the apatite mineral phase. In addition, evaluation of protein secondary structure in the 35-day-old rat femur from the Amide I and Amide II vibrations near 1650 and 1550 cm-1, respectively, revealed that protein conformation was altered by ethanol fixation.

Amides↗
PubMed1992

FT-IR microscopic mappings of early mineralization in chick limb bud mesenchymal cell cultures.

Chick limb bud mesenchymal cells differentiate into chondrocytes and form a cartilaginous matrix in culture. In this study, the mineral formed in different areas within cultures supplemented with 4 mM inorganic phosphate, or 2.5, 5.0, and 10 mM beta-glycerophosphate (beta GP), was characterized by Fourier-transform infrared (FT-IR) microscopy. The relative mineral-to-matrix ratios, and distribution of crystal sizes at specific locations throughout the matrix were measured from day 14 to day 30. The only mineral phase detected was a poorly crystalline apatite. Cultures receiving 4 mM inorganic phosphate had smaller crystals which were less randomly distributed around the cartilage nodules than those in the beta GP-treated cultures. beta GP-induced mineral consisted of larger, more perfect apatite crystals. In cultures receiving 5 or 10 mM beta GP, the relative mineral-to-matrix ratios (calculated from the integrated intensities of the phosphate and amide I bands, respectively) were higher than in the cultures with 4 mM inorganic phosphate or in the in vivo calcified chick cartilage.

Animals↗
PubMed1992

Acyl chain conformational ordering in 1,2 dipalmitoylphosphatidylethanolamine. Integration of FT-IR and 2H NMR results.

The extent of trans-gauche isomerization at the 4 and 4' positions of the acyl chains of fully hydrated 4,4,4',4'-d4 1,2-dipalmitoylphosphatidylethanolamine (4-d4 DPPE) bilayers was quantitatively evaluated from the infrared (IR) intensity of the CD2 rocking modes. About 20% gauche conformers were observed at 72 degrees C (above Tm), while at 23 degrees C, well below Tm, about 4% were noted. The order parameter SC-D was determined from 2H nuclear magnetic resonance (NMR) quadrupolar splittings. SC-D is the product of a segmental order parameter (S gamma), which depends on conformational order, and a chain order parameter (S alpha) which depends on slower motions such as chain wobble. The IR-determined percentage of gauche forms was converted into a segmental order parameter and factored out of the measured value for SC-D to yield an estimate of S alpha = 0.59 for L alpha phase DPPE. A comparison with S alpha for 1,2-dipalmitoylphosphatidylcholine (DPPC) suggests that increased wobble is responsible for enhanced motional averaging of the quadrupolar splittings in the latter at a similar reduced temperature. The extent of conformational disordering [at the 4(4') position] is essentially unchanged between the two molecules. The current study demonstrates the advantage of integrating quantitative IR with 2H NMR data, for elucidation of the contributions of the individual motions that average the NMR quadrupolar splittings.

Deuterium↗
PubMed1992

An infrared study of the interaction of polymethyl methacrylate with the protein and mineral components of bone.

We used Fourier transform infrared (FT-IR) microscopic mapping techniques to investigate the infiltration of polymethyl methacrylate (PMMA), a widely used medium for embedding biological tissues, into rat femur sections. Monitoring of the infrared absorbances of the PMMA carbonyl stretch, the protein amide I, and the apatite mineral phosphate stretch over a 225 x 975-microns region of the epiphyseal growth plate region of the rat femur enabled comparison of the relative amount of each component in distinct regions of the tissue. It was found that PMMA penetrates less into regions of greater mineral density and that the frequency of the PMMA carbonyl absorbance from the embedded tissue, 1729 cm-1, is identical to the free PMMA carbonyl frequency. This is consistent with a diffusion mechanism of infiltration of the PMMA, with no specific chemical interaction between the PMMA and the tissue components.

Animals↗
PubMed1991

Fourier transform infrared studies of secondary structure and orientation of pulmonary surfactant SP-C and its effect on the dynamic surface properties of phospholipids.

SP-C, a highly hydrophobic, 3.7-kDa protein constituent of lung surfactant, has been isolated from bovine lung lavage, purified, and reconstituted into binary lipid mixtures of 1,2-dipalmitoyl-phosphatidylcholine (DPPC) and 1,2-dipalmitoylphosphatidylglycerol (DPPG). Fourier transform infrared (FT-IR) spectroscopy has been applied to examine SP-C secondary structure, the average orientation of alpha-helical segments relative to the bilayer normal in membrane films, and the effect of protein on the thermotropic properties of the phospholipid acyl chains. In addition, dynamic surface measurements were made on phospholipid films at the A/W interface in the presence and absence of SP-C. SP-C (0.5 mol %) was found to possess about 60% alpha-helical secondary structure in lipid vesicles. Higher levels (1.5 mol %) of SP-C resulted in a slight increase of beta-forms, possibly resulting from protein aggregation. The helical segments exhibited an average angle of orientation of about 24 degrees with respect to the bilayer normal, suggesting a trans-bilayer orientation of the peptide. The observation that 70% of the peptide bond hydrogens are hard to exchange in D2O further reflects the hydrophobic nature of the molecule. SP-C produced little effect on the thermotropic properties of the binary lipid mixture, as measured from acyl chain C-H and C-D stretching frequencies. However, the presence of 1 mol % protein markedly reduced the viscance and increased the elasticity of surface films suggesting a mechanism by which SP-C facilitates the spreading of phospholipids on an aqueous surface. The possible physiological consequences of these observations are discussed.

Amino Acid Sequence↗
PubMed1991

CD2 rocking modes as quantitative infrared probes of one-, two-, and three-bond conformational disorder in dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylcholine/cholesterol mixtures.

The use of CD2 rocking modes in the IR spectrum as quantitative probes of phospholipid conformational disorder has recently been described for aqueous dispersions of 1,2-dipalmitoylphosphatidylcholine (DPPC) and DPPC/cholesterol mixtures [Mendelsohn et al. (1989) Biochemistry 28, 8934-8939; Davies et al. (1990) Biochemistry 29, 4368-4373]. Initial studies focused at the 4, 6, and 10 acyl chain positions of DPPC. In the current work, the method is extended to the 2, 3, 12, and 13 positions. Conformational disorder in the L alpha phase is approximately the same (about 20% gauche) at positions 4, 10, and 13, but an unexpected higher value is observed (about 30%) at the 6 position. Cholesterol (33 mol%) restricts gauche rotamer formation by factors ranging from 6 to 9 at positions 4 and 6, respectively, to 1.5-2 at positions 10, 12, and 13. Quantitative analysis for the DPPC/cholesterol "liquid-ordered" phase indicates the occurrence of 1.2 gauche bonds/chain, a marked reduction from the 3.6-4.2 gauche bonds/chain for DPPC alone. Proximity to the ester moiety at acyl chain position 3 perturbs the vibrational coupling patterns of the CD2 rocking modes and eliminates their sensitivity to conformational change. In addition, the feasibility of a method based on the conformation-dependent coupling between CD2 rocking frequencies of two successive CD2 groups for the quantitative detection of specific, position-dependent king (gtg') and isolated gauche (gtt) conformers is demonstrated. Finally, comparisons between IR measurements and explicit theoretical predictions of acyl chain conformational order are presented.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1991

Novel infrared spectroscopic method for the determination of crystallinity of hydroxyapatite minerals.

Biologically important apatite analogues have been examined by Fourier Transform Infrared Spectroscopy (FT-IR), and a method developed to quantitatively assess their crystalinity. Changes in the phosphate v1 and v3 regions, 900-1,200 cm-1, for a series of synthetic (containing hydroxide, fluoride, or carbonate ion) and biological apatites with crystal sizes of 100-200 A were analyzed with curve-fitting and second derivative spectroscopy. The v1,v3 contour was composed of three main subbands. Correlations were noted between two spectral parameters and crystal size as determined by x-ray diffraction. The percentage area of a component near 1,060 cm-1 decreased as the length of the c-axis of the hydroxyapatite (HA) compounds increased, while the frequency of a band near 1,020 cm-1 increased with increasing length of the apatite c-axis. These parameters are thus proposed as indices of crystallinity for biological (poorly crystalline) HA. The FT-IR spectra of highly crystalline apatitic compounds were also analyzed. For crystal sizes of 200-450 A, the percentage area of the phosphate v1 band (near 960 cm-1) decreased with increasing HA crystal size. IR indices of crystallinity have thus been developed for both well crystallized and poorly crystallized HA derivatives. The molecular origins of the various contributions to the v1,v3 contour are discussed, and a preliminary application of the method to a microscopic biological sample (rat epiphyseal growth plate) is illustrated.

Crystallography↗
PubMed1990

Effects of cholesterol on conformational disorder in dipalmitoylphosphatidylcholine bilayers. A quantitative IR study of the depth dependence.

A method originally proposed by Snyder and Poore [(1973) Macromolecules 6, 708-715] as a specific probe of trans-gauche isomerization in hydrocarbon chains and recently applied [Mendelsohn et al. (1989) Biochemistry 28, 8934-8939] to the quantitative determination of phospholipid acyl chain conformational order is utilized to monitor the effects of cholesterol at various depths in dipalmitoylphosphatidylcholine (DPPC) bilayers. The method is based on the observation that the CD2 rocking modes from the acyl chains of specifically deuterated phospholipids occur at frequencies in the Fourier transform infrared spectrum which depend upon the local geometry (trans or gauche) of the C-C-C skeleton surrounding a central CD2 group. Three specifically deuterated derivatives of DPPC, namely, 4,4,4',4'-d4 DPPC (4-d4 DPPC), 6,6,6',6'-d4 DPPC (6-d4 DPPC), and 12,12,12',12'-d4 DPPC (12-d4 DPPC), have been synthesized, and the effects of cholesterol addition at 2:1 DPPC/cholesterol (mol:mol) on acyl chain order at various temperatures have been determined. At 48 degrees C, cholesterol inhibits gauche rotamer formation by factors of approximately 9 and approximately 6 at positions 6 and 4, respectively, of the acyl chains, thus demonstrating a strong ordering effect in regions of the bilayer where the sterol rings are presumed to insert parallel to the DPPC acyl chains. In contrast, the ability of the sterol to order the acyl chains is much reduced at the 12-position. The sterol demonstrates only a slight disordering of phospholipid gel phases. Finally, the contributions of different classes of gauche conformers to the spectra have been determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholesterol↗
PubMed1990

A quantitative infrared determination of acyl chain conformation in gramicidin/dipalmitoylphosphatidylcholine mixtures.

A quantitative infrared characterization of phospholipid acyl chain disordering in 6,6,6'6'-d4 dipalmitoylphosphatidylcholine/ Gramicidin D bilayers has been made. Three CD2 rocking modes, at 622 cm-1, 646-649 cm-1, and 651-653 cm-1, assigned to particular conformers, were used to determine disorder in the presence of peptide, as well as percentages of particular classes of conformer within the total gauche population. At 44C, the gauche percentages in 10:1 and 30:1 lipid/peptide mixtures were 15% and 17%, respectively. At 34C, the corresponding values were 9.8% and 2.6%. The percentage of (single gauche bend + kink) conformers, relative to multiple gauche forms, decreases dramatically from 78% in the 30:1 mixture to 15% in the 10:1 mixture at 44C. These data provide the first quantitative measure of the extent to which a membrane-spanning peptide disorders phospholipid gel phases and orders liquid crystal phases.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1990

Effects of cholesterol on the interaction of Ca2(+)-ATPase with 1-palmitoyl-2-oleoylphosphatidylethanolamine. An FTIR study.

Ca2(+)-ATPase from rabbit skeletal muscle has been isolated, purified, and reconstituted into vesicles containing binary mixtures of 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE)/cholesterol. Fourier transform infrared spectroscopy (FTIR) was used to investigate the effect of protein on the thermotropic behavior of POPE in these reconstituted ternary complexes. The CH2 symmetric stretching modes of the phospholipid acyl chains near 2850 cm-1 served as an index of the melting process. The thermotropic transition of the POPE component in a 103:12:1 (POPE/cholesterol/Ca2(+)-ATPase) complex was shifted to lower temperatures compared with a protein-free binary lipid mixture of the same relative proportions. When combined with differential scanning calorimetric (DSC) data for the binary (POPE/cholesterol) lipid systems, this observation suggests that Ca2(+)-ATPase preferentially sequesters 15-35 molecules of POPE from the lipid mixture and therefore excludes cholesterol from its immediate environment. Higher levels of cholesterol in ternary complexes progressively eliminate the cooperative POPE melting event.

Animals↗
PubMed1990

The interviewer's "presenting problems" in the initial interview.

Complex countertransference responses that contribute to the interviewer's presenting problems in the initial interview with a patient are related to: the clinician's preinterview fantasies, the interviewee's empirical reality, and the patient as experienced creatively. Presenting problems may be an unavoidable component of clinical contact, but when the interviewer responds to them with psychological-mindedness, they can contribute to realistic assessment and positive case disposition.

Countertransference↗
PubMed1989

Quantitative determination of conformational disorder in the acyl chains of phospholipid bilayers by infrared spectroscopy.

A method is proposed and demonstrated for the direct determination of conformational disorder (trans-gauche isomerization) as a function of acyl-chain position in phospholipid bilayer membranes. Three specifically deuterated derivatives of dipalmitoylphosphatidylcholine (DPPC), namely 4,4,4',4'-d4-DPPC (4-d4-DPPC), 6,6,6',6'-d4-DPPC (6-d4-DPPC), and 10,10,10',10'-d4-DPPC (10-d4-DPPC), have been synthesized. The CD2 rocking modes in the Fourier transform infrared (FT-IR) spectrum have been monitored as a function of temperature for each derivative. A method originally applied by Snyder and Poore [(1973) Macromolecules 6, 708-715] as a specific probe of hydrocarbon chain conformation in alkanes has been used to analyze the data. The rocking modes appear at 622 cm-1 for a CD2 segment surrounded by a trans C-C-C skeleton and between 645 and 655 cm-1 for segments surrounded by particular gauche conformers. The integrated band intensities of these modes have been used to monitor trans-gauche isomerization in the acyl chains at particular depths in the bilayer. At 48 degrees C, above the gel-liquid-crystal phase transition, the percentage of gauche rotamers present is 20.7 +/- 4.2, 32.3 +/- 2.3, and 19.7 +/- 0.8 for 4-d4-DPPC, 6-d4-DPPC, and 10-d4-DPPC, respectively. The gel phase of the latter two molecules is highly ordered. In contrast, a substantial population of gauche rotamers was observed for the 4-d4-DPPC. The conformational analysis yields a range of 3.6-4.2 gauche rotamers/acyl chain of DPPC above the phase transition. This range is in excellent accord with the dilatometric data of Nagle and Wilkinson [(1978) Biophys. J. 23, 159-175]. The significant advantages of the FT-IR approach are discussed.

Circular Dichroism↗
PubMed1989

Fourier-transform infrared spectroscopy studies of lipid/protein interaction in pulmonary surfactant.

The thermotropic behavior of intact bovine lung surfactant and its hydrophobic extract has been monitored via the temperature dependence of the 2850 cm-1 phospholipid acyl chain CH2 symmetric stretching frequencies in the IR spectrum. A broad, reversible, melting event was noted from about 15 to 40 degrees C in both the lipid extract and the native surfactant. Slight protein-induced disordering of the lipid acyl chains was evident. The melting event was confirmed by differential scanning calorimetry. The major surfactant protein, a 30-36-kDa class of glycoprotein (SP-A), has been isolated from bovine lung lavage and purified by affinity chromatography. SP-A was reconstituted into a binary lipid mixture of acyl chain perdeuterated dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol (DPPC-d62/DPPG, 85:15 w/w), a ratio which approximates that in surfactant. Use of DPPC-d62 permitted the FT-IR determination of the effect of protein on the thermotropic behavior of individual phospholipids in the binary mixture. High levels of SP-A induced an ordering of the phospholipids, as shown by an increase in the transition temperature of DPPC-d62 compared to the lipid model. In contrast, a mixture of the other surfactant proteins induced a progressive disordering of the phospholipids and disruption of the cooperativity of the melting event. Transition widths of about 3 degrees, 9 degrees, and 27 degrees were noted for protein:lipid ratios of 0, 1:1, and 2:1 (w/w), respectively. Possible roles for the various proteins in surfactant function are discussed in light of these data.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1989

FT-IR microscopy of endochondral ossification at 20 mu spatial resolution.

A Fourier transform infrared spectrometer has been coupled with an optical microscope to study the distribution and characteristics of the mineral phase in calcifying tissues at 20 mu spatial resolution. This represents the first biophysical application of this technique. High quality spectra were obtained in a relatively short scan time (1-2 minutes) from thin longitudinal sections of normal and rachitic rat femurs. Substantial spatial variations in the extent and structure of the mineral phase were observed as a function of spatial position both within and beyond the growth plates, as judged by the phosphate vibrations in the 900-1200 cm-1 spectral region. The current experiments reveal the utility of FT-IR microscopy in identification of sites where mineralization has occurred. In addition to vibrations from the inorganic components, the Amide I and Amide II motions of the protein constituents are readily observed and may be useful as a probe of protein/mineral interactions.

Animals↗
PubMed1989

Infrared spectroscopic investigations of pulmonary surfactant. Surface film transitions at the air-water interface and bulk phase thermotropism.

The molecular structure of the phospholipid component of intact pulmonary surfactant isolated from bovine lung lavage has been examined by Fourier transform infrared spectroscopy. Two different physical states of the surfactant were examined by means of different infrared spectroscopic sampling techniques. Transmission infrared experiments were used to study the surfactant in the bulk phase. In these experiments, the thermotropic behavior of the bulk surfactant was monitored by temperature-induced variations in the phospholipid acyl chain CH2 stretching frequencies. A broad phase transition (confirmed by differential scanning calorimetry) was noted with an onset temperature near 15 degrees C and a completion temperature near 42 degrees C. In addition to the bulk transmission experiments, external reflection infrared spectroscopy was used to examine surfactant films in situ at the air-water interface. As surface pressure was increased from 0 to 43 dyn/cm, a gradual and continuous decrease in the CH2 stretching frequency was noted for the surfactant. Thus, under surface pressures which correspond to large lung volumes in vivo, the surfactant acyl chains exist mostly in the ordered (trans) configuration. The frequency shift in the CH2 stretching mode is consistent with a continuous ordering of the acyl chains upon compression over the pressure range 0-43 dyn/cm, and implies that a weakly cooperative phase transition occurs in the hydrocarbon region of the surface film. The surface film transition is especially noted in the pressure-area curve of the surfactant and approximates in two dimensions the broad thermotropic phase transition of the bulk phase surfactant. Substantial differences were observed between the response to surface pressure changes of intact surfactant compared with the main surfactant phospholipid, 1,2-dipalmitoyl-sn--glycero-3-phosphocholine. The changes in response are attributed to the presence of additional surfactant components. The current work demonstrates the ability of infrared spectroscopy to obtain structural information on the surfactant in physical states that directly relate to those in vivo.

1,2-Dipalmitoylphosphatidylcholine↗