Spin-label studies on phosphatidylcholine-cholesterol membranes: effects of alkyl chain length and unsaturation in the fluid phase

Dynamic properties of phosphatidylcholine-cholesterol membranes in the fluid phase and water accessibility to the membranes have been studied as a function of phospholipid alkyl chain length, saturation, mole fraction of cholesterol, and temperature by using spin and fluorescence labelling methods. The results are the following: (1) The effect of cholesterol on motional freedom of 5-doxyl stearic acid spin label (5-SASL) and 16-doxyl stearic acid spin label (16-SASL) in saturated phosphatidylcholine membrane is significantly larger than the effects of alkyl chain length and introduction of unsaturation in the alkyl chain. (2) Variation of alkyl chain length of saturated phospholipids does not alter the effects of cholesterol except in the case of dilauroylphosphatidylcholine, which possesses the shortest alkyl chains (12 carbons) used in this work. (3) Unsaturation of the alkyl chains greatly reduces the ordering effect of cholesterol at C-5 and C-16 positions although unsaturation alone gives only minor fluidizing effects. (4) Introduction of 30 mol% cholesterol to dimyristoylphosphatidylcholine membranes decreases the lateral diffusion constants of lipids by a factor of four, while it causes only a slight decrease of lateral diffusion in dioleoylphosphatidylcholine membranes. (5) If compared at the same temperature, 5-SASL mobilities plotted as a function of mole fraction of cholesterol in the fluid phases of dimyristoylphosphatidylcholine-, dipalmitoylphosphatidylcholine- and distearoylphosphatidylcholine-cholesterol membranes are similar in wide ranges of temperature (45-82 degrees C) and cholesterol mole fraction (0-50%). (6) In isothermal experiments with saturated phosphatidylcholine membranes, 5-SASL is maximally immobilized at the phase boundary between Regions I and III reported by other workers (Recktenwald, D.J. and McConnell, H.M. (1981) Biochemistry 20, 4505-4510) and becomes more mobile away from the boundary in Regions I and III. (7) 5-SASL in unsaturated phosphatidylcholine membranes showed a gradual monotonic immobilization with increase of cholesterol mole fraction without showing any maximum in the range of cholesterol fractions studied. (8) By rigorously determining rigid-limit magnetic parameters of cholestane spin labels in membranes from Q-band second-derivative ESR spectra to monitor the dielectric environment around the nitroxide radical, it is concluded that cholesterol incorporation increases water accessibility in the hydrophilic loci of the membrane. In contrast, 12-(9-anthroyloxy)stearic acid fluorescence showed that water accessibility is decreased in the hydrophobic loci of the membrane.     

The Expression of Race-Specific Resistance of Wheat Seedlings to Puccinia recondita

A range of wheat cultivars with resistance factors effective against Puccinia recondita f. sp. tritici was studied to investigate the mode of action and expression of resistance at first and third seedling leaf stages. In most cultivars, resistance to isolate 74/2 resulted in extremely low levels of infection, apparently linked with a predominantly hypersensitive response by the host. In seedlings of cultivars Mans Fundin and Sterna, however, race-specific resistance was expressed as increased pathogen latent period and intermediate infection levels.     

Decarboxylation kinetics for the carbonatotris(pyridine)cobalt(III) ion in perchloric acid solutions

The kinetics of the decomposition reactions of the CO(py)₃(CO₃)(H₂O)⁺ ion have been investigated in aqueous perchloric acid solutions over a range of hydrogen ion concentrations (0.10 to 5.0 M) and at two ionic strengths (I = 1.0 and 5.0 M). At the lower ionic strength, plots of ln (A_t–A_∞ versus time show a nonlinearity that is consistent with that expected for consecutive first-order reactions. The rates of the faster reaction are similar to those reported for the spontaneous reduction of aquopyridine-cobalt(III) cations. At the higher ionic strength, the above noted curvature is not apparent and the decarboxylation kinetics of the title complex may be described by a pseudo-first-order rate constant: k_obs = k[H₃O⁺]. At 20°C, k = (1.75−+0.09) s⁻¹ M⁻¹ with activation parameters ofΔH^‡ = (97 −+ 4) kJ mol⁻¹ and ΔS^‡ = −(54 −+ 32) J deg⁻¹ mol⁻¹. These kinetic parameters are compared with those previously reported for the similar complexes, Co(py)₄CO₃⁺ and Co(py)₂(CO₃)(H₂O)₂⁺.     

Regional differences in quantities of histochemically detectable mucosubstances in nasal, paranasal, and nasopharyngeal epithelium of the bonnet monkey

Inhaled irritants induce secretory cell hyperplasia in nasal epithelium of animals. To characterize this response histochemically it is first important to know the histochemical character and distribution of epithelial mucosubstance in the normal nasal cavity. An automated image analyzing method was used to detect and quantitate acidic, neutral, and sulfated mucosubstances in the epithelium lining the nasal and paranasal airways of eight bonnet monkeys. Tissue sections 2 micron thick from defined regions of these airways were stained with either alcian blue/periodic acid-Schiff to demonstrate acid and neutral mucosubstances or high iron diamine to demonstrate sulfated mucins. Respiratory epithelium covering maxilloturbinates had the largest volume of stainable mucosubstance per unit surface area of basal lamina, whereas the maxillary sinus epithelium had the least. There was a general anteroposterior increase in the quantity of total epithelial mucosubstance along the septal and lateral walls of the nasal cavity, and there was more acidic than neutral mucosubstance in the posterior nasal airway than in the anterior. Epithelial mucosubstance in the maxillary sinus was predominantly neutral. Therefore, we conclude that there are substantial regional quantitative differences in stainable mucosubstances in the primate nasal epithelium which must be considered when examining nasal mucosa for irritant-induced changes in epithelial mucins.     

Interactions of 14N:15N stearic acid spin-label pairs: effects of host lipid alkyl chain length and unsaturation

Electron-electron double resonance (ELDOR) and saturation recovery electron paramagnetic resonance (EPR) spectroscopy have been employed to examine the interactions of 14N:15N stearic acid spin-label pairs in fluid-phase model membrane bilayers composed of a variety of phospholipids. The [14N]-16-doxylstearate:[15N]-16-doxylstearate (16:16) pair was utilized to measure lateral diffusion of the spin-labels, while the [14N]-16-doxylstearate:[15N]-5-doxylstearate (16:5) pair provided information on vertical fluctuations of the 16-doxylstearate nitroxide moiety toward the membrane surface. Three saturated host lipids of varying alkyl chain length [dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC)], an alpha-saturated, beta-unsaturated lipid [1-palmitoyl-2-oleoylphosphatidylcholine (POPC)], and phosphatidylcholine from a natural source [egg yolk phosphatidylcholine (egg PC)] were utilized as host lipids. Lateral diffusion of the stearic acid spin-labels was only slightly affected by alkyl chain length at a given reduced temperature (Tr) in the saturated host lipids but was significantly decreased in POPC at the same Tr. Lateral diffusion in DMPC, POPC, and egg PC was quite similar at 37 degrees C. A strong correlation was noted between lateral diffusion constants and rotational mobility of [14N]-16-doxylstearate. Vertical fluctuations were likewise only slightly influenced by alkyl chain length but were strongly diminished in POPC and egg PC relative to the saturated systems. This diminution of the 16:5 interaction was observed even under conditions where no differences were discernible by conventional EPR. These studies indicate that vertical fluctuation of 16-doxylstearate is quite sensitive to host lipid unsaturation and that ELDOR studies of interactions between 14N:15N spin-label pairs can provide information on spin-label motion beyond that given by conventional EPR.     

Correlation of polarized absorption spectroscopic and X-ray diffraction studies of crystalline cytosolic aspartate aminotransferase of pig hearts

Absorption spectra of large, well-formed crystals of cytosolic aspartate aminotransferase have been recorded using plane polarized light. Making use of measurements of crystal thickness we have calculated extinction coefficients with the electric vector of the light parallel to both the a and c axes of the crystals of the enzyme in space group P2(1)2(1)2(1). The spectra have been resolved into components with lognormal distribution curves and the resulting integrated intensities have been used to calculate the c/a polarization ratios for the absorption bands of the bound co-enzyme pyridoxal 5'-phosphate. From the polarization ratio and the co-ordinates of the co-enzyme ring atoms, provided by X-ray crystallography, we have assigned principal molecular directions of the transition dipole moment within the plane of the co-enzyme ring. Of two possible orientations, only one predicts the correct crystal extinction coefficients for the 436 nm band. In this orientation, when viewed from the B face of the ring (i.e. looking into the active site of the enzyme), the transition moment is related to the N-1-C-4 axis of the ring by counterclockwise rotation by 27 degrees. A tentative assignment of the principal molecular directions of the transition moment has also been made for the 368 nm band of the high pH form of the enzyme. In each case, the plane of the co-enzyme ring was located from the atomic co-ordinates of the ring atoms and of those atoms attached directly to the ring. The projection of the N-1 to C-4 axis on to this plane was used to evaluate the orientation of the transition moment, which was presumed to lie precisely within the plane of the ring. We have tilted this plane systematically to evaluate the error in transition moment direction resulting from uncertainties in the atomic co-ordinates. When 2-methylaspartate is diffused into the crystals if forms a Schiff base with the co-enzyme in which the ring has tilted about 32 degrees from its original position and the polarization ratio of the 436 nm band drops from 1.6 in the free enzyme to about 0.38. On the assumption that the orientation of the transition moment within the co-enzyme does not change during this rotation, this value of the polarization ratio is within experimental error of that predicted from X-ray structures on the two forms. The 2-methylaspartate binds only to subunit 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Examination of bile acid negative feedback regulation in rats

Recent data obtained using cultured rat hepatocytes showed that bile acids do not inhibit bile acid synthesis, whereas cholesterol concentrations vary in parallel with bile acid synthesis (Davis et al. (1983. J. Biol. Chem. 258: 4079-4082). This led us to re-evaluate in vivo experiments upon which the consensus that bile acid synthesis is primarily regulated by bile acid "negative feedback" is based. Infusion of taurocholate into either the jugular vein or duodenum of bile-diverted rats stimulated biliary cholesterol secretion and bile flow, but it did not inhibit bile acid synthesis. The lack of an inhibitory effect was evident using several different infusion rates of taurocholate. Even at the greatest rate of taurocholate infusion (25 mumol/(100 g.hr] there was no significant inhibition of bile acid synthesis. In contrast, infusing mevinolin (1 mg/hr), a potent competitive inhibitor of HMG-CoA reductase, almost completely inhibited bile acid synthesis and biliary cholesterol secretion. Since mevinolin did not affect bile flow, these results cannot be ascribed to bile secretory failure. Thus, while these studies suggest that taurocholate may not regulate bile acid synthesis directly via negative feedback, cholesterol is likely to act as a positive effector of bile acid synthesis.     

Morphogenesis of the respiratory bronchiole in rhesus monkey lungs

The epithelium of the respiratory bronchiole in the adult rhesus monkey consists of two populations: a pseudostratified epithelium with basal, mucous goblet, and ciliated cells located near the pulmonary artery (PA); and a simple cuboidal epithelium composed only of nonciliated bronchiolar epithelial (or Clara) cells in areas away from the PA. This study describes the pattern of differentiation of these two epithelial populations, and their relationship to the PA and to the time of appearance of alveoli in the respiratory bronchiole of the rhesus monkey during the period of 90-125 days gestational age (DGA). These events were related to changes in the adjacent parenchyma. Dissected airways of infusion-fixed, critical-point-dried lungs were evaluated by scanning microscopy followed by light microscopy of the same airways. At 54% of gestation (90 DGA), the distal airway was lined by a mixture of ciliated and nonciliated cells. By 67% of gestation (110 DGA), the ciliated cells were confined to the epithelium over the PA. The underlying connective tissue initially was cellular containing few fibers but was fibrous by 76% of gestation (125 DGA). Alveolarization began near the most distal cartilage at 57% of gestation (95 DGA), the same period at which secondary septation occurred in the distal acinus. Thus, alveolarization occurred simultaneously in two centers: 1) the proximal centriacinar region in the vicinity of the most distal cartilage and 2) the distal lung parenchyma. The duration of centriacinar alveolarization was short, approximately 5 days.     

Comparative development of thoracic intervertebral discs and intra-articular ligaments in the human, monkey, mouse, and cat

Developmental features of thoracic intervertrebral discs and their association in the adult with other vertebral structures were investigated in four species. The human anulus fibrosus, nucleus pulposus, and intra-articular ligaments were compared to those of the fetal rhesus monkey, mouse, and kitten. Photomicrographs of transverse sections of intervertebral discs document the presence of intra-articular ligaments in fetuses of these four species. Both transverse and sagittal sections of kittens were used to identify the intercapital ligament as it differentiated from the dorsal part of the intra-articular ligament. Relatively frequent dorsal herniation of the thoracic nucleus pulposus in humans may be due to the vestigial nature of the human intra-articular ligament. Quadrupeds have well-developed intra-articular ligaments, which explains anatomically the paucity of dorsal protrusions of the nucleus pulposus into the vertebral canal in the thoracic region of the cat and mouse when compared to the human. The intra-articular ligament was closely associated with the developing prenatal mammalian intervertebral disc in the four species studied, and this relationship and its surgical importance are described.