A Fourier transform infrared spectroscopic study of the molecular interaction of cholesterol with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine

The temperature dependencies of the infrared spectra of pure and cholesterol-containing multibilayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine were studied using Fourier transform infrared techniques. A comparison of the spectroscopic data showed the retention of a melting phenomenon at 60 mol% cholesterol content, and the retention of some all-trans conformations in the liquid-crystalline phase. It is also demonstrated that at temperatures less than 30 degrees C, the cholesterol-containing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine multibilayers still contain a small amount of pure 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, packed in an orthorhombic subcell lattice. Spectral changes were found in the absorptions characteristic of the phospholipid head groups. The addition of cholesterol results in changes in the ester bands, and demonstrates the induction by cholesterol of non-equivalent ester conformations.     

The phase transition of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine as seen by Fourier transform infrared difference spectroscopy

The potential of Fourier transform infrared difference spectroscopy for biochemical applications is demonstrated by the gel to liquid crystal phase transition of the title compound. While the changes occurring in the vibrational pattern of the hydrophobic palmitoyl chains are easily monitored, this technique also discriminates between no change in the choline moiety and a small yet significant change in the carbonyl moiety, both located in the hydrophylic head group.     

Pressure-temperature gelatinization phase diagram of starch: An in situ Fourier transform infrared study

The gelatinization of rice starch is reported as a function of temperature and pressure from the changes in the ir spectrum. The diagram that is observed is reminiscent of those obtained for the denaturation of proteins and the phase separation observed from the cloud point for several water soluble synthetic polymers. It is proposed that the reentrant shape of the diagram for starch is not only due to hydrogen bonding but also to the imperfect packing of amylose and amylopectin chains in the starch granule. The influence of pressure and temperature on thermodynamic parameters leading to this diagram is discussed.     

Fourier transform infrared study of the halorhodopsin chloride pump

Halorhodopsin (hR) is a light-driven chloride pump located in the cell membrane of Halobacterium halobium. Fourier transform infrared difference spectroscopy has been used to study structural alterations occurring during the hR photocycle. The frequencies of peaks attributed to the retinylidene chromophore are similar to those observed in the spectra of the related protein bacteriorhodopsin (bR), indicating that in hR as in bR an all-trans----13-cis isomerization occurs during formation of the early bathoproduct. Spectral features due to protein structural alterations are also similar for the bR and hR photocycles. For example, formation of the red-shifted primary photoproducts of both hR and bR results in similar carboxyl peaks in the 1730-1745-cm-1 region. However, in contrast to bR, no further changes are observed in the carboxyl region during subsequent steps in the hR photocycle, indicating that additional carboxyl groups are not directly involved in chloride translocation. Overall, the close similarity of vibrations in hR and bR photoproduct difference spectra supports the existence of some common elements in the molecular mechanisms of energy transduction and active transport by these two proteins.     

Fourier transform infrared spectroscopic identification of gel phase domains in reconstituted phosphilipid vesicles containing Ca2+-ATPase

Ca²⁺-ATPase from rabbit sarcoplasmic reticulum has been isolated, purified, and reconstituted into lipid environments containing as primary components 1,2-dielaidoylphosphatidylcholine (DEPC) and acyl-chain perdeuterated 1,2-dimyristoylphosphatidylcholine (DMPC-d₅₄). Differential scanning calorimetry (DSC) has been used to elucidate the phase behavior of this lipid pair while Fourier transform infrared spectroscopy (FT-IR) has been used to monitor the state of each lipid component in the presence of protein. The lipid mixture shows gel state miscibility over at least most of the composition range, a result in good accord with Van Dijck et al. (Biochim. Biophys. Acta 470, 58–69 (1977)), for the binary mixture with proteated DMPC. Acyl chain perdeuteration thus does not greatly alter the miscibility properties of the lipid pair. Reconstitution of Ca²⁺-ATPase with this lipid pair proceeds with moderate efficiency. Up to 80% of the endogenous lipid can be replaced depending on the lipid composition. Unusual composition-dependent protein-induced effects on lipid melting properties are noticed. At low levels of DMPC-d₅₄, both the DEPC and DMPC-d₅₄ components have their melting processes broadened and shifted to lower temperatures, compared with binary lipid mixtures of the same composition. This suggests that protein perturbs both lipids in similar fashion. At high levels of DMPC-d₅₄, the DEPC component exhibits a highly cooperative melting process at temperatures close to that for pure DEPC. This strongly indicates that domains of DEPC are present (at least at low temperatures) in the bilayer, and that Ca²⁺-ATPase is excluded from these domains. The protein thus exhibits preferential interaction with the DMPC-d₅₄ component. This work demonstrates the utility of FT-IR for identification of the molecular origin of particular domains in reasonably complex lipid mixtures. The relevance of this work to native membrane systems where lipid domains have been observed by several groups is discussed.     

Differential scanning calorimetric and Fourier transform infrared spectroscopic investigations of cerebroside polymorphism

Calorimetric and Fourier transform infrared (FTIR) spectroscopic studies have been made of the polymorphism exhibited by bovine brain cerebroside-water systems, and the effect of cholesterol and dipalmitoylphosphatidylcholine (DPPC) upon this polymorphism was investigated. The conversion of the cerebroside from the thermodynamically stable to the metastable form is found to be accompanied by spectral changes, indicating a decrease in cerebroside headgroup hydration and a rearrangement of the hydrogen-bond network. The incorporation of low concentrations of cholesterol and DPPC into cerebroside bilayers broadens the thermal transitions associated with the cerebroside as a result of the disruption of cerebroside-cerebroside interactions. This disruption is evident in the spectra of cerebroside/cholesterol mixtures.     

Fourier transform infrared spectroscopic identification of gel phase domains in reconstituted phospholipid vesicles containing Ca2+-ATPase

Ca2+-ATPase from rabbit sarcoplasmic reticulum has been isolated, purified, and reconstituted into lipid environments containing as primary components 1,2-dielaidoylphosphatidylcholine (DEPC) and acyl-chain perdeuterated 1,2-dimyristoylphosphatidylcholine (DMPC-d54). Differential scanning calorimetry (DSC) has been used to elucidate the phase behavior of this lipid pair while Fourier transform infrared spectroscopy (FT-IR) has been used to monitor the state of each lipid component in the presence of protein. The lipid mixture shows gel state miscibility over at least most of the composition range, a result in good accord with Van Dijck et al. (Biochim. Biophys. Acta 470, 58-69 (1977)), for the binary mixture with proteated DMPC. Acyl chain perdeuteration thus does not greatly alter the miscibility properties of the lipid pair. Reconstitution of Ca2+-ATPase with this lipid pair proceeds with moderate efficiency. Up to 80% of the endogenous lipid can be replaced depending on the lipid composition. Unusual composition-dependent protein-induced effects on lipid melting properties are noticed. At low levels of DMPC-d54, both the DEPC and DMPC-d54 components have their melting processes broadened and shifted to lower temperatures, compared with binary lipid mixtures of the same composition. This suggests that protein perturbs both lipids in similar fashion. At high levels of DMPC-d54, the DEPC component exhibits a highly cooperative melting process at temperatures close to that for pure DEPC. This strongly indicates that domains of DEPC are present (at least at low temperatures) in the bilayer, and that Ca2+-ATPase is excluded from these domains. The protein thus exhibits preferential interaction with the DMPC-d54 component. This work demonstrates the utility of FT-IR for identification of the molecular origin of particular domains in reasonably complex lipid mixtures. The relevance of this work to native membrane systems where lipid domains have been observed by several groups is discussed.     

Fourier transform infrared study of the N intermediate of bacteriorhodopsin

Visible absorption spectroscopic experiments show that the N intermediate is the main photoproduct of a highly hydrated film of the light-adapted bacteriorhodopsin (70% water by weight) at pH 10 and 274 K. The difference Fourier transform infrared spectrum between the N intermediate and unphotolyzed light-adapted bacteriorhodopsin was recorded under these conditions. A small amount of the M intermediate present did not affect this spectrum significantly. The difference spectrum exhibited a positive band at 1755 cm-1 (probably due to Asp-85) and a negative band at 1742 cm-1 (due to Asp-96), neither of which was observed for the M intermediate. The spectrum of the N intermediate at pH 7 was nearly identical with that at pH 10. Spectra at pH 10 also were measured with isotope-substituted samples. A vibrational band at 1692 cm-1 due to the peptide bond disappeared, and a band at 1558 cm-1 emerged upon formation of the N intermediate. The spectrum also displayed bands containing the N-H and C15-H in-plane bending vibrational modes at 1394 and 1303 cm-1. These frequencies are similar to those of the L intermediate while the intensities of these bands are larger than those in the L intermediate, suggesting that the Schiff bases of both the L and N intermediates have a strong hydrogen-bonding interaction with the protein and that the C12-H to C15-H region of the chromophore is less twisted in the N intermediate than in the L intermediate.     

Interaction of 7,7,8,8-tetracyanoquinodimethane with diacylphosphatidylcholines and -phosphatidylglycerols. A photoacoustic Fourier transform infrared study

7,7,8,8-Tetracyanoquinodimethane (TCNQ) was incorporated in fully hydrated liposomes of the following pyrene-containing as well as non-labelled phospholipids: 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphatid ylc holine (PPDPC), 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphatidyl- rac'- glycerol (rac'-PPDPG), 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphatidyl- sn-3'- glycerol (3'-PPDPG), 1-[10-(pyren-1-yl)decanoyl]-2-palmitoyl-sn-glycero-3-phosphatidyl- sn-3'- glycerol (3'-PDPPG), 1-[10-pyren-1-yl)decanoyl]-2-palmitoyl-sn-glycero-3-phosphatidyl-s n-1'- glycerol (1'-PDPPG), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidyl-rac'-glycerol (rac'-DPPG). Lyophilized charge-transfer (CT) complexes of TCNQ with phospholipids were examined by Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS). Due to the spectral changes observed in the vibrational bands originating from the CH2 and C = O stretching vibrations, and the bands associated with the polar headgroup of the phospholipids it is evident that TCNQ has only a minor perturbing effect on the hydrocarbon chains. However, the molecular interaction between TCNQ and phospholipids is seen in the polar headgroup region. The donated electrons are most likely located on the oxygens of the phosphate group in the polar head. As judged from the present infrared data interactions of TCNQ with phosphatidylcholines (PC) and phosphatidylglycerols (PG) differ. For PG the complex formation produces a second strong C = O stretching band at approx. 1710 cm-1 in addition to the band at approx. 1735 cm-1 indicating a specific molecular interaction in the interfacial region.     

Fourier transform infrared study of proteins with parallel beta-chains

Deconvolved and second derivative Fourier transform infrared spectra of the proteins flavodoxin and triosephosphate isomerase have been obtained in the 1600 to 1700 cm-1 (amide I) region. To our knowledge these results provide the first experimental infrared data on proteins with parallel beta-chains. Characteristic absorption bands for the parallel beta-segments are observed at 1626-1639 cm-1 (strong) and close to 1675 cm-1 (weak). Previous theoretical studies based on hypothetical models with large, regular beta-sheets had suggested bands close to 1650 and 1666 cm-1. Our new assignments were confirmed by band area measurements, which yield conformational information in good agreement with results from X-ray diffraction data. The spectra were compared with corresponding spectra of concanavalin A and carboxypeptidase A. The first contains only antiparallel beta-segments, the second "mixed" beta-segments, with some strands lying antiparallel and others parallel. None of the observed amide I band frequencies assigned to parallel beta-chains occurs in the 1650 cm-1 region associated with helical segments.     

Lipid phase transitions measured in intact cells with Fourier transform infrared spectroscopy

Lipid phase transitions in membranes are thought to be a major damaging event during cooling of cells prior to cryopreservation or during warming after freeze-thaw has been completed. Although there is abundant evidence that such transitions occur in isolated phospholipids, the evidence that they are found in membranes in intact cells is less clear, due largely to technical difficulties in detecting such transitions in the complex mixtures of lipids and proteins found in natural membranes. We show here that Fourier transform infrared spectroscopy provides a rapid, convenient method for detecting these transitions in intact cells. We have used intact pollen grains of cattail (Typha latifolia) as a primary experimental subject. Spectra taken of the intact pollen grains show most of the features commonly seen in natural membrane vesicles or pure phospholipids. Shifts in the vibrational frequency and width of the CH2 bands with temperature can be used to detect lipid phase transitions. Biochemical analysis, coupled with the spectroscopy, was used to assign transitions to nonpolar and polar lipids. Finally, although assignment of the melting lipid unambiguously in other cells has not yet been made, we show that the transitions can nevertheless be detected in other intact cells, including those of four plant species and sperm of three animals.     

A Difference Fourier transform infrared study of tyrosyl radical Z* decay in photosystem II.

Photosystem II (PSII) contains a redox-active tyrosine, Z* Difference Fourier transform infrared (FTIR) spectroscopy can be used to obtain structural information about this species, which is a neutral radical, Z*, in the photooxidized form. Previously, we have used isotopic labeling, inhibitors, and site-directed mutagenesis to assign a vibrational line at 1478 cm(-1) to Z*; these studies were performed on highly resolved PSII preparations at pH 7.5, under conditions where Q(A)(-) and Q(B)(-) make no detectable contribution to the vibrational spectrum (Kim, Ayala, Steenhuis, Gonzalez, Razeghifard, and Barry. 1998. Biochim. Biophys. Acta. 1366:330-354). Here, time-resolved infrared data associated with the reduction of tyrosyl radical Z* were acquired from spinach core PSII preparations at pH 6.0. Electron paramagnetic resonance spectroscopy and fluorescence control experiments were employed to measure the rate of Q(A)(-) and Z* decay. Q(B)(-) did not recombine with Z* under these conditions. Difference FTIR spectra, acquired over this time regime, exhibited time-dependent decreases in the amplitude of a 1478 cm(-1) line. Quantitative comparison of the rates of Q(A)(-) and Z* decay with the decay of the 1478 cm(-1) line supported the assignment of a 1478 cm(-1) component to Z*. Comparison with difference FTIR spectra obtained from PSII samples, in which tyrosine is labeled, supported this conclusion and identified other spectral components assignable to Z* and Z. To our knowledge, this is the first kinetic study to use quantitative comparison of kinetic constants in order to assign spectral features to Z*.     

A low-temperature structural phase transition of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine bilayers in the gel phase

A new thermotropic phase transition, at ?30°C and atmospheric pressure, was found to occur in the gel phase of aqueous DPPC dispersions. The Raman spectral changes at this phase transition are similar to those observed in the gel phase of DMPC dispersions at ?60°C. The thermotropic phase transition at ?30°C is equivalent to the barotropic GII to GIII phase transition observed in DPPC at 1.7 kbar and 30°C. It is shown that the rate of the large angle interchain reorientational fluctuations decreases gradually with decreasing temperature, and that the orientationally disordered acyl chain structure of the GII phase is extended into the GIII phase of DPPC. The interchain interaction, arising from the damping of the reorientational fluctuations, increases with decreasing temperature in the GII gel phase as well as in the GIII gel phase.     

Structural and functional relationships in 5'-nucleotidase from bull seminal plasma. A Fourier transform infrared study.

Fourier transform infrared spectroscopy (FTIR) was used to investigate the secondary structure of 5'-nucleotidase from bull seminal plasma (BSP). Spectra of protein in both D2O and H2O were analyzed by deconvolution and second derivative methods in order to observe the overlapping components of the amide I band. The protein, which is made up of two apparently identical subunits and which contains two zinc atoms, was studied in its native form, in the presence of dithiotreitol (DTT) and after removal of the two zinc atoms by means of nitrilotriacetic acid (NTA). Deconvolved and second derivative spectra of amide I band showed that the native protein contains mostly beta-sheet structure with a minor content of alpha-helix. The quantitative analysis of the amide I components was performed by a curve-fitting procedure which revealed 54% beta-sheet, 18% alpha-helix, 22% beta-turns and 6% unordered structure. The second derivative and deconvolved spectra of amide I band showed that no remarkable changes in the secondary structure of 5'-nucleotidase were induced by either DTT or NTA. These results were confirmed by the curve-fitting analysis where little or no changes occurred in the relative content of amide I components when the protein was treated with DTT or with NTA. Major changes, however, were observed in the thermal denaturation behavior of the protein. The native protein showed denaturation at temperatures between 70 and 75 degrees C, while the maximum of denaturation was observed between 65 and 70 degrees C and between 55 and 60 degrees C in the presence of NTA and DTT, respectively. The results obtained indicate that the two separate subunits of the protein have essentially the same secondary structure as that of the native enzyme.     

Fourier transform infrared analysis of bacteriorhodopsin secondary structure.

Fourier transform infrared spectroscopy at a resolution of 1 cm-1 has been used to study the conformation of dark-adapted bacteriorhodopsin in the native purple membrane, in H2O and D2O suspensions. A detailed analysis of the amide I bands was made using derivative and deconvolution techniques. Curve-fitting results of four independent experiments indicate, after estimation of the methodological errors, that native bacteriorhodopsin contains 52-73% alpha-helices, 13-19% reverse turns, 11-16% beta-sheets, and 3-7% unordered segments. Our analysis has enabled the identification of several components corresponding to alpha-helices, beta-sheets, and reverse turns. Besides the alpha I- and alpha II-helices (peaking at 1658 and 1665 cm-1), we propose that two more infrared bands arise from alpha-helical structures: one at 1650 cm-1 from alpha I and another one at 1642 cm-1 in H2O suspension, which could originate from type III beta-turns (i.e., one turn of 3(10)-helix). The relatively high content of reverse turns suggests the presence of one reverse turn per loop, plus another one in the C-terminal segment. On the other hand, several reasons argue that the calculated mean beta-sheet content of around 14% should be decreased somewhat. These beta-sheets could be located in the noncytoplasmatic links of the bacteriorhodopsin molecule.     

Tyrosine protonation changes in bacteriorhodopsin. A Fourier transform infrared study of BR548 and its primary photoproduct

The structural alterations which occur in bacteriorhodopsin (bR) during dark adaptation (BR570----BR548) and the primary phototransition of the dark photocycle (BR548----KD610) have been investigated by Fourier transform infrared and UV difference spectroscopy. Possible contributions of tyrosine to the Fourier transform infrared difference spectra of these transitions were assigned by incorporating ring per-deuterated tyrosine into bR. Based on these data and UV difference measurements, we conclude that a stable tyrosinate exists in BR570 at physiological temperature and that it protonates during formation of BR548. A tyrosinate protonation has also been observed at low temperature during the primary phototransition of BR570 to the red-shifted photoproduct K630 (1). However, we now find that no tyrosine protonation change occurs during the primary phototransition of BR548 to the red-shifted intermediate KD610. Through analysis of bR containing isotopically labeled retinals, it was also determined that the chromophore of KD610 exits in a 13-trans, 15-cis configuration. On the basis of this evidence and previous studies on the structure of the chromophore in BR570, BR548, and K630, it appears that only the 13-trans,15-trans configuration of the protonated chromophore leads to a stable tyrosinate group. It is proposed that a tyrosinate residue is stabilized due to its interaction with the Schiff base positive charge in the BR570 chromophore. Isomerization of the chromophore about either the C13 = C14 or C = N bond disrupts this interaction causing a protonation of the tyrosinate.     

Studies of mixed-chain diacyl phosphatidylcholines with highly asymmetric acyl chains: a Fourier transform infrared spectroscopic study of interfacial hydration and hydrocarbon chain packing in the mixed interdigitated gel phase.

The mixed interdigitated gel phases of unlabeled, specifically 13C = O-labeled, and specifically chain-perdeuterated samples of 1-O-eicosanoyl, 2-O-lauroyl phosphatidylcholine and 1-O-decanoyl, 2-O-docosanoyl phosphatidylcholine were studied by infrared spectroscopy. Our results suggest that at the liquid-crystalline/gel phase transition temperatures of these lipids, there is a greater redistribution in the populations of free and hydrogen-bonded ester carbonyl groups than is commonly observed with symmetric chain n-saturated diacyl phosphatidylcholines. The formation of the mixed interdigitated gel phase coincides with the appearance of a marked asymmetry in the contours of the C = O stretching band, a process which becomes more pronounced as the temperature is reduced. This asymmetry is ascribed to the emergence of a predominant lipid population consisting of free sn1- and hydrogen-bonded (hydrated) sn2-ester carbonyl groups. This suggests that the region of the mixed interdigitated bilayer polar/apolar interface near to the sn1-ester carbonyl group is less hydrated than is the case with the noninterdigitated gel-phase bilayers formed by normal symmetric chain phosphatidylcholines. In the methylene deformation region of the spectrum, the unlabeled lipids exhibit a pronounced splitting of the CH2 scissoring bands. This splitting is significantly attenuated when the short chains are perdeuterated and collapses completely upon perdeuteration of the long chains, irrespective of whether the long (or short) chains are esterified to the sn1 or sn2 positions of the glycerol backbone. These results are consistent with a global hydrocarbon chain packing motif in which the zigzag planes of the hydrocarbon chains are perpendicular to each other and the sites occupied by long chains are twice as numerous as those occupied by short chains. The experimental support for this chain-packing motif enabled more detailed considerations of the possible ways in which these lipid molecules are assembled in the mixed interdigitated gel phase. Generally, our results are compatible with a previously proposed model in which the mixed interdigitated gel phase is an assembly of repeat units which consists of two phosphatidylcholine molecules forming a triple-chain structure with the long chains traversing the bilayer and with the methyl termini of the shorter chains opposed at the bilayer center. Our data also suggest that the packing format which is most consistent with our results and previously published work is one in which the hydrocarbon chains of each repeat unit are parallel to each other with the repeat units themselves being perpendicularly packed.     

Secondary structure and orientation of the surfactant protein SP-B in a lipid environment. A Fourier transform infrared spectroscopy study.

Attenuated total reflection Fourier transform infrared spectroscopy was used to investigate the secondary structure of the surfactant protein SP-B. Nearly half of the polypeptide chain is folded in an alpha-helical conformation. No significant change of the secondary structure content was observed when the protein is associated to a lipid bilayer of dipalmitoylphosphatidylcholine (DPPC)/phosphatidylglycerol (PG) or of dipalmitoylphosphatidylglycerol (DPPG). The parameters related to the gamma w(CH2) vibration of the saturated acyl chains reveal no modification of the conformation or orientation of the lipids in the presence of SP-B. A model of orientation of the protein at the lipid/water interface is proposed. In this model, electrostatic interactions between charged residues of SP-B and polar headgroups of PG, and the presence of small hydrophobic alpha-helical peptide stretches slightly inside the bilayers, would maintain SP-B at the membrane surface.     

Ionization state of the coenzyme 5'-phosphate ester in cytosolic aspartate aminotransferase. A Fourier transform infrared spectroscopic study

In order to determine the ionization state of the 5'-phosphate of bound pyridoxal phosphate, a Fourier transform infrared spectroscopic study of cytosolic aspartate aminotransferase has been carried out. Dianionic and monoanionic phosphate monoesters give rise to two bands each in the infrared spectrum [Shimanouchi, T., Tsuboi, M., & Kyogoku, Y. (1964) Adv. Chem. Phys. 8, 435-498]. These bands can be identified in infrared spectra of the free coenzyme in solution. Due to interfering bands arising from the protein, only the band assigned to the symmetric stretching of the dianionic phosphate is observed in holoenzyme solutions. The integrated intensity of this band does not change with pH in the range 5.3-8.6, while for free pyridoxal phosphate, the integrated intensity of the same band changes with pH according to the pK value expected for the 5'-phosphate group in solution. Moreover, the value of the integrated intensity for the bound cofactor is close to the value given by free cofactor at pH 8-9. These results suggest that the 5'-phosphate of the bound cofactor remains mostly dianionic throughout the investigated pH range and disfavor other interpretations in terms of ionization of the phosphate group on the basis of the nuclear magnetic resonance 31P chemical shift-pH titration curve of holoenzyme [Schnackerz, K. D. (1984) in Chemical and Biological Aspects of Vitamin B6 Catalysis (Evangelopoulos, E. A., Ed.) Part A, pp 195-208, Alan R. Liss, New York].(ABSTRACT TRUNCATED AT 250 WORDS)