Mechanical and chemical injury to thylakoid membranes during freezing in vitro

The relative contributions of membrane rupture due to osmotic stress and of chemical membrane damage due to the accumulation of cryotoxic solutes to cryoinjury was investigated using thylakoid membranes as a model system. When thylakoid suspensions were subjected to a freeze-thaw cycle in the presence of different molar ratios of NaCl as the cryotoxic solute and sucrose as the cryoprotective solute, membrane survival first increased linearly with the osmolality of the solutions used to suspend the membranes, regardless of the molar ratio of salt to sucrose. It subsequently decreased when the ratio of sucrose to salt was not sufficiently high for complete cryopreservation by sucrose. There was an optimum of cryopreservation at intermediate osmolalities (approx. 0.1 osmol/kg). This optimum of cryopreservation at a given sucrose concentration could be shifted to lower solute concentration, if mixtures of NaCl and NaBr were used instead of NaCl alone. At suboptimal initial osmolalities, damage is attributed mainly to membrane rupture. Under these conditions, cryopreservation is not influenced by the chaotropicity of the suspending medium. At supraoptimal initial solute concentrations, solute (i.e., chemical) effects determine membrane survival. Under these conditions, increased ratios of sugar to salt increased cryoprotection. In mixtures of NaCl and NaBr at constant molar ratios of salt to sucrose, chemical membrane damage was quantitatively related to the lyotropic properties of the ions used. The degree of chemical damage becomes more pronounced with rising osmolalities of the suspending media. With NaF as the cryotoxic solute, damage was more severe than should be expected from its lyotropic properties. This may reflect a specific interaction of fluoride with the membranes. Protein release from the membranes during freezing in the presence of different anions was qualitatively comparable at identical ratios of sugar to salt. However, the total amount of protein released was correlated linearly with membrane inactivation, even when different anions acted on the membranes. Gel electrophoretic analysis of proteins released from thylakoid membranes during freezing revealed discrete bands indicative of mechanical and chemical damage, respectively.     

The effects of ozone on growth and stomatal response in the F<Subscript>2</Subscript> generation of hybrid poplar (<Emphasis Type="Italic">Populus trichocarpa × Populus deltoides</Emphasis>)

Thirty-six F₂ hybrid poplar (Populus trichocarpa × P. deltoides) clones were fumigated with ozone to record its effects on growth, correlate them with stomatal response and screen for ozone sensitivity. Fumigation was applied for 6 to 9 h each day for approximately 3 months at ozone concentrations of 85 to 128 μg g⁻¹ using open-top chambers. Height, diameter, number of leaves, stomatal conductance, transpiration rate, total biomass, biomass components and root/shoot ratios were reduced by ozone stress. Percent of leaf fall in ozone-treated plants was nearly three times higher than in control plants exposed to charcoal-filtered air. Leaf senescence, because of ozone exposure, did not appear to be associated with reduced biomass production. Some clones had a high percentage of leaf-fall with ozone exposure, but were able to maintain total biomass production near that of the control. Their response may be an example of an ability to adjust or compensate for ozone damage. There was no significant or consistent relationship between stomatal conductance and total biomass or the change in stomatal conductance as a result of ozone exposure and the change in total biomass. Taken together, these results suggest that effects of ozone on poplar growth cannot be solely correlated to changes in stomatal conductance, more physiological and biochemical parameters should be examined.     

臭氧分布及对呼吸系统的影响

臭氧是氧气的同素异形体,在常温条件下它是一种有特殊臭味的淡蓝色气体。臭氧现已成为我国主要的一种环境污染物。臭氧的分布存在着明显的时间特异性和地区特异性,与气象条件、污染物的输送与扩散以及局地光化学反应强度等因素有着重要的关系。同时在人群流行病学和动物实验中发现了臭氧可以提高慢性阻塞性肺疾病、哮喘等呼吸系统疾病的发病率和病死率,同时提示了老年人、有呼吸系统疾病病史的人群和儿童都是易受臭氧影响的高危人群。国内外学者也对臭氧对机体损伤的机制进行了研究,主要集中在臭氧的致炎作用、氧化应激作用、造成机体气道高反应性和对细胞DNA的损伤等方面。本文主要就国内外对臭氧在时空分布、对机体呼吸系统影响和臭氧造成机体损伤的机制这3方面作一简要综述。     

Evaluation of the antiulcerogenic activity of violacein and its modulation by the inclusion complexation with beta-cyclodextrin

The effects of beta-cyclodextrin (betaCD) inclusion complexation on the ability of violacein to prevent gastric ulceration in mice were studied. Violacein-betaCD inclusion complexes were prepared in 1:1 and 1:2 molar ratios and analysed by differential scanning calorimetry and powder X-ray diffractometry. Violacein previously administered orally at 10 mg/kg significantly reduced indomethacin-induced gastric lesions, as well as 100 mg/kg of cimetidine (positive control). However, betaCD complexation in both molar ratios significantly potentiated the protective action of violacein. In the HCl--ethanol-induced gastric ulcer model, violacein and the 1:2 inclusion complex (10 mg/kg, p.o.) inhibited gastric damage by almost 85%, whereas a 63% reduction was observed for the positive control, lansoprazole, at 30 mg/kg. In contrast, treatment with the 1:1 inclusion complex resulted in almost total disappearance of the antiulcer activity in this model. No significant changes in stress-induced gastric injury were found. In addition, the 1:2 inclusion complex improved the antilipoperoxidant activity of violacein in rat liver cells exposed to t-butyl hydroperoxide, whereas the 1:1 complex was less active than violacein. In summary, the 1:2 betaCD inclusion complex has gastroprotective properties similar to or higher than that of violacein. An increase in mucosal defensive mechanisms and protection against peroxidative damage might be involved.     

Selection of a biopolymer based on attachment,morphology and proliferation of fibroblast NIH/3T3 cells for the development of a biodegradable tissue regeneration template: Alginate,bacterial cellulose and gelatin

Material selection is one of the most important aspects to construct a novel template for tissue regeneration. In this research alginate, bacterial cellulose (BC) and gelatin were selected based on available material and published data to prepare membranes and then the morphology of Swiss mouse embryo fibroblast NIH/3T3 cells on the surface of these membranes was examined to select the best material for the development of a biodegradable skin tissue regeneration template. The cells on alginate membrane crosslinked with Ca²⁺ (AGM_Ca) showed a spherical morphology and growth retardation, probably due to high calcium content on and in the surface of membrane. This has been confirmed in control experiments in which calcium was added to the culture medium (DMEM medium). The NIH/3T3 cells grown on the BC membrane (BC_M) and membrane of glutaraldehyde (GTA) crosslinked gelatin (GTA_GM) had a polygonal morphology. The proliferation rate of cells on the GTA_GM was faster than that on the BC_M. Therefore the GTA_GM is better than the AGM_Ca and BC_M to apply as a skin tissue regeneration template. Cytotoxic effects of GTA were studied in GTA_GMs prepared using gelatins obtained from cow bones, pork skins, and fish skins. It was found that molar ratio of GTA to gelatin for preparation of membrane should not be higher than 0.033. Cytotoxic effects of GTA were observed on the GTA_GMs prepared with molar ratio higher than 0.033, except pork skin gelatin membrane with a molar ratio, 0.033, which showed the cytotoxic effects on fibroblast cells. The physical morphology of the membranes of cow bone gelatin and fish skin gelatin is stronger and more flexible than that of pork skin gelatin in wet forms. According to these results, it can be suggested that pork skin gelatin might have had less crosslinked points, NH₂-sites of lysine molecules than cow bone gelatin and fish skin gelatin. These two gelatins are selected for further development of a template for skin tissue regeneration.     

The interaction of phosphatidylcholine bilayers with Triton X-100

The interaction of multilamellar phosphatidylcholine vesicles with the non-ionic detergent Triton X-100 has been studied under equilibrium conditions, specially in the sub-lytic range of surfactant concentrations. Equilibrium was achieved in less than 24 h. Estimations of detergent binding to bilayers, using [3H]Triton X-100, indicate that the amphiphile is incorporated even at very low concentrations (below its critical micellar concentration); a dramatic increase in the amount of bound Triton X-100 occurs at detergent concentrations just below those producing membrane solubilization. Solubilization occurs at phospholipid/detergent molar ratios near 0.65 irrespective of lipid concentration. The perturbation produced by the surfactant in the phospholipid bilayer has been studied by differential scanning calorimetry, NMR and Fourier-transform infrared spectroscopy. At low detergent concentration (lipid/detergent molar ratios above 3), a reduction in 2H-NMR quadrupolar splitting occurs, suggesting a decrease in the static order of the acyl chains; the same effect is detected by Fourier-transform infrared spectroscopy in the form of blue shifts of the methylene stretching vibration bands. Simultaneously, the enthalpy variation of the main phospholipid phase transition is decreased by about a third with respect to its value in the pure lipid/water system. For phospholipid/detergent molar ratios between 3 and 1, the decrease in lipid static order does not proceed any further; rather an increase in fluidity is observed, characterized by a marked decrease in the midpoint transition temperature of the gel-to-fluid phospholipid transition. At the same time an isotropic component is apparent in both 31P-NMR and 2H-NMR spectra, and a new low-temperature endotherm is detected in differential scanning calorimetric traces. When phospholipid and Triton X-100 are present at equimolar ratios some bilayer structure persists, as judged from calorimetric observations, but NMR reveals only one-component isotropic signals. At lipid/detergent molar ratios below unity, the NMR lines become narrower, the main (lamellar) calorimetric endotherm tends to vanish and solubilization occurs.     

Intermolecular contact between globular N-terminal fold and C-terminal domain of ApoA-I stabilizes its lipid-bound conformation: studies employing chemical cross-linking and mass spectrometry

The structure of apoA-I on discoidal high density lipoprotein (HDL) was studied using a combination of chemical cross-linking and mass spectrometry. Recombinant HDL particles containing 145 molecules of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and two molecules of apoA-I with a 96-A diameter were treated with the lysine-specific cross-linker, dithiobis(succinimidylpropionate) at varying molar ratios from 2:1 to 200:1. At low molar ratios of dithiobis(succinimidylpropionate) to apoA-I, two products were obtained corresponding to approximately 53 and approximately 80 kDa. At high molar ratios, these two products merged, yielding a product of approximately 59 kDa, close to the theoretical molecular mass of dimeric apoA-I. To identify the intermolecular cross-links giving rise to the two different sized products, bands were excised from the gel, digested with trypsin, and then analyzed by liquid chromatography-electrospray-tandem mass spectrometry. In addition, tandem mass spectrometry of unique cross-links found in the 53- and 80-kDa products suggested that a distinct conformation exists for lipid-bound apoA-I on 96-A recombinant HDL, emphasizing the inherent flexibility and malleability of the N termini and its interaction with its C-terminal domain.     

Bilirubin induces loss of membrane lipids and exposure of phosphatidylserine in human erythrocytes

Unconjugated bilirubin increasingly binds to erythrocytes as the bilirubin-to-albumin molar ratio exceeds unity, leading to toxic manifestations that can culminate in cell lysis. Our previous studies showed that bilirubin induces the release of lipids from erythrocyte membranes. In the present work, those studies were extended in order to characterize the alterations of membrane lipid composition and evaluate whether bilirubin leads to a loss of phospholipid asymmetry. To this end, human erythrocytes were incubated with several bilirubin-to-albumin molar ratios (0.5 to 5), and cholesterol as well as the total and the individual classes of phospholipids were determined. To detect erythrocytes with phosphatidylserine at the outer surface, the number of annexin V-positive cells was determined following incubation with bilirubin, fixing its molar ratio to albumin at 3. The results demonstrate profound changes in erythrocyte membrane composition, including modified cholesterol and phospholipid content. The release of membrane cholesterol, as well as of total and individual classes of phospholipids at molar ratios ≥1, indicates that damage of erythrocytes may occur in severely ill jaundiced neonates. The loss of the inner-located phospholipids, phosphatidylethanolamine and phosphatidylserine, points to a redistribution of phospholipids in the membrane bilayer. This was confirmed by the exposure of phosphatidylserine at the outer cell surface. In conclusion, this study demonstrates that bilirubin induces loss of membrane lipids and externalization of phosphatidylserine in human erythrocytes. These features may facilitate hemolysis and erythrophagocytosis, thus contributing to enhanced bilirubin production and anemia during severe neonatal hyperbilirubinemia. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genotoxic effect of ozone in human peripheral blood leukocytes

The genotoxic effect of ozone was studied in human leukocytes in vitro, using the single cell gel electrophoresis (SCGE) assay. Cell treatment for 1 h at 37 degrees C with 0.9-5.3 mM O(3) resulted in a dose-dependent increase of DNA damage, comparable to that induced by 4-40 mM of H(2)O(2), used as a positive control. This effect of ozone was reversed by post-treatment incubation of the cells for 45-90 min at 37 degrees C, and prevented by pre-incubation of the cells with catalase (20 microg/ml). These results demonstrate that O(3) induces DNA-damage in primary human leukocytes. The damage is rapidly repaired, and probably mediated by the formation of H(2)O(2).     

The Effect of Calcium on Non-heme Iron Uptake,Efflux, and Transport in Intestinal-like Epithelial Cells (Caco-2 Cells)

It has been suggested that calcium inhibits the absorption of dietary iron by directly affecting enterocytes. However, it is not clear if this effect is due to a decreased uptake of iron or its efflux from enterocytes. We studied the effect of calcium on the uptake, efflux, and net absorption of non-heme iron using the intestinal-like epithelial cell line Caco-2 as an in vitro model. Caco-2 cells were incubated for 60 min in a buffer supplemented with non-heme iron (as sulfate) and calcium to achieve calcium to iron molar ratios ranging from 50:1 to 1,000:1. The uptake, efflux, and net absorption of non-heme iron were calculated by following a radioisotope tracer of ⁵⁵Fe that had been added to the buffer. Administration of calcium and iron at molar ratios between 500 and 1,000:1 increased the uptake of non-heme iron and decreased efflux. Calcium did not have an effect on the net absorption of non-heme iron. At typical supplementary doses for calcium and non-heme iron, calcium may not have an effect on the absorption of non-heme iron. The effect of higher calcium to iron molar ratios on the efflux of non-heme iron may be large enough to explain results from human studies.     

Differential effects induced by α- and β-endosulfan in lipid bilayer organization are reflected in proton permeability

The effects of two insecticides isomers, α- and β-endosulfan, on the passive proton permeability of large unilamellar vesicles (LUV) reconstituted with dipalmitoylphosphatidylcholine (DPPC) or mitochondrial lipids were reported. In DPPC (LUV) gel phase, at 30 °C, the global kinetic constant (K) of proton permeability (proportional to the proton permeability) initially increased slightly with the increase of α-endosulfan/lipid molar ratio up to 0.143. In the range from 0.143 to 0.286, a discontinuity in the increment occurred and, above this range, the proton permeability increased substantially. In DPPC fluid phase, at 48 °C, the proton permeability showed a behavior identical to that observed in gel DPPC, with a sharp increase for α-endosulfan/lipid molar ratios ranging from 0.143 to 0.286. At these and higher concentrations, α-endosulfan induced phase separation in the plane of DPPC membranes, as revealed by differential scanning calorimetry (DSC). Conversely to α-endosulfan, β-endosulfan induced only a slight increase in the proton permeability, either in the fluid or the gel phase of DPPC, for all β-endosulfan/lipid molar ratios tested. Additionally, the effects of the endosulfan isomers on the proton permeability of mitochondrial fluid lipid dispersions, at 37 °C, are similar to those described for DPPC. The β-isomer induced a very small effect, and α-endosulfan, at low concentrations, increased slightly the proton permeability, but for insecticide/lipid molar ratios above 0.143 the permeability increased substantially. Consequently, the membrane physical state of synthetic and native lipid dispersions, as affected by the structural features of α- and β-endosulfan, influenced the proton permeability. The effects here observed in vitro suggest that the formation of lateral membrane domains may underlay the biological activity of α-endosulfan in vivo, contributing to its higher degree of toxicity as compared with β-endosulfan.     

DNA degradation with ozone

DNA was ozonized in solution and the reaction was followed with polarimetry and with iodimetry. Polarimetry was used to determine the molar ratio DNA/O₃ when the DNA optical activity vanishes completely. At a molar ratio DNA/O₃ = 2.3 the supramolecular structure of DNA collapses completely. Instead, iodimetry shows that the ozonolysis proceeds until all the nucleobases have been destroyed, an event which occurs at a molar ratio DNA/O₃ = 1.1. The ozonolysis of DNA was also followed spectrophotometrically.

DNA is reactive with ozone also in the solid state, as fixed bed. Clear indication about its oxidation derives from the FT-IR spectra from polarimetric measurements and from thermal analysis performed by thermogravimetric analysis (TGA), differential thermogravimetric analysis (DTG) and from differential thermal analysis (DTA).

Particular remarkable is the fact that RNA has been found much less reactive toward ozone in the solid state than DNA.     

Apoptosis induced by ozone and oxysterols in human alveolar epithelial cells

The mechanism of ozone-induced lung cell injury is poorly understood. One hypothesis is that ozone induces lipid peroxidation and that these peroxidated lipids produce oxidative stress and DNA damage. Oxysterols are lipid peroxides formed by the direct effects of ozone on pulmonary surfactant and cell membranes. We studied the effects of ozone and the oxysterol 5β,6β-epoxycholesterol (β-epoxide) and its metabolite cholestan-6-oxo-3,5-diol (6-oxo-3,5-diol) on human alveolar epithelial type I-like cells (ATI-like cells) and type II cells (ATII cells). Ozone and oxysterols induced apoptosis and cytotoxicity in ATI-like cells. They also generated reactive oxygen species and DNA damage. Ozone and β-epoxide were strong inducers of nuclear factor erythroid 2-related factor 2, heat shock protein 70, and Fos-related antigen 1 protein expression. Furthermore, we found higher sensitivity of ATI-like cells compared to ATII cells exposed to ozone or treated with β-epoxide or 6-oxo-3,5-diol. In general the response to the cholesterol epoxides was similar to the effect of ozone. Understanding the response of human ATI-like cells and ATII cells to oxysterols may be useful for further studies, because these compounds may represent useful biomarkers in other diseases.     

Human serum albumin and its structural variants mediate cholesterol efflux from cultured endothelial cells

In the present study, we used the human EA.hy926 endothelial cell line as the model system to investigate the effect of human serum albumin (HSA) and its structural variants on cholesterol efflux. Initial studies showed that HSA promoted cholesterol efflux in a dose- and time-dependent manner, reaching a plateau at 10 mg/ml at 90 min. As a control, gelatin displayed no significant effect on efflux, while HSA was significantly more efficient than ovalbumin and bovine serum albumin (BSA) in promoting cholesterol efflux. Equal molar concentrations of HSA and apolipoprotein A-I (apoA-I) showed that apoA-I had considerably higher efficiency in efflux. However, the prevailing high plasma concentrations of HSA may compensate for its lower efflux rate compared to apoA-I. To characterize the mechanism of HSA-mediated cholesterol efflux, we studied the effects of cAMP and temperature on efflux using both EA.hy926 endothelial cells and murine RAW 264.7 macrophages. We found that HSA-mediated efflux occurred via a cAMP-independent and relatively temperature-insensitive pathway. We next examined the nature of HSA-cholesterol interaction by comparing the effects of various HSA mutants to wild-type HSA on cholesterol efflux. We found specific interactions between subdomains 2A and 3A and cholesterol, as indicated by the changes in the efflux rate of various HSA mutants. In conclusion, our study provides evidence for the role of HSA in cholesterol efflux, and shows that the substitution of specific amino acid residues in subdomains of 2A and 3A may be important structural determinants in its ability to bind to cholesterol and participate in cholesterol efflux.     

Contributions of the MMP-2 collagen binding domain to gelatin cleavage. Substrate binding via the collagen binding domain is required for hydrolysis of gelatin but not short peptides.

Two matrix metalloproteinases, MMP-2 and MMP-9, contain each three fibronectin type II-like modules, which form their collagen binding domains (CBDs). The contributions of CBD substrate interactions to the catalytic activities of these gelatinases have attracted special interest. Recombinant (r) CBDs retain collagen binding properties and deletions of CBDs in these MMPs reduce activities on collagen and elastin. We have characterized further the requirement of the CBD for MMP-2 cleavage of gelatin. The analyses used intact rMMP-2 and rCBD to eliminate any confounding effects that might result from structural perturbations in rMMP-2 induced by deletion of the approximately 20 kDa internal CBD. In protein-protein binding assays, 2% DMSO disrupted gelatin interactions of both rCBD and rMMP-2. At this concentration, DMSO also reduced the gelatinolytic activity by approximately 70%, pointing to a central role of CBD-substrate interactions during MMP-2 cleavage of gelatin. Subsequently, soluble rCBD was determined to competitively inhibit gelatin binding of unmodified rMMP-2 to gelatin by 73% and to reduce the MMP-2 degradation of gelatin by 70-80%. The residual gelatin cleavage that was not inhibited even by molar excess rCBD could be accounted for by degradation of short substrate molecules. Indeed, rCBD inhibited rMMP-2 cleavage of an 11 amino acid collagen-like peptide substrate (NFF-1) by less than 10%. These observations were confirmed with enzyme extracts from experimental tumors in mice. In the presence of rCBD, approximately 65% of the MMP-derived gelatinolytic activity was eliminated. Together, these results demonstrate that the CBD is absolutely required for MMP-2 cleavage of full-length collagen alpha-chains, but not for short protein fragments such as those generated by hydrolysis of gelatin.     

Effect of gelatin on molecular mobility in amorphous sucrose detected by erythrosin B phosphorescence

We have used phosphorescence from the triplet probe erythrosin B (Ery B) to evaluate the effect of gelatin on the molecular mobility of the amorphous sucrose matrix as a function of temperature. Ery B was dispersed in amorphous sucrose and sucrose-gelatin films at ratios of approximately 1:10(4) (probe/sucrose), and delayed emission spectra and emission decay transients were measured over the temperature range from 5 to 100 degrees C. Analysis of spectra using a lognormal function provided the peak energy and bandwidth of the emission. The emission peak frequency decreased at low (0.00022-0.0007) gelatin concentrations and increased at high (above 0.0022) gelatin concentrations, indicating that gelatin increased the extent, and thus the rate, of dipolar relaxation at low gelatin content and decreased the extent at higher gelatin content. Decay transients were well fit to a stretched exponential function at all gelatin contents and temperatures. Analysis of the emission lifetimes provided a measure of the rate of non-radiative decay to the ground state, an indicator of matrix molecular mobility. This rate increased at low (0.00022-0.0022) and decreased at high (>0.0073) gelatin wt ratios. Analysis of the effect of gelatin on the emission bandwidth, the stretching exponent beta, and the variation of lifetime across the emission band indicated that matrix dynamic site heterogeneity increased at low and decreased at high gelatin wt ratios. These results provide a novel insight into the complex dynamic effects of the gelatin polymer on the molecular mobility of the amorphous sucrose matrix.     

Ozone Quenching Properties of Isoprene and Its Antioxidant Role in Leaves

Isoprene is formed in and emitted by plants and the reason for this apparent carbon waste is still unclear. It has been proposed that isoprene stabilizes cell and particularly chloroplast thylakoid membranes. We tested if membrane stabilization or isoprene reactivity with ozone induces protection against acute ozone exposures. The reduction of visible, physiological, anatomical, and ultrastructural (chloroplast) damage shows that clones of plants sensitive to ozone and unable to emit isoprene become resistant to acute and short exposure to ozone if they are fumigated with exogenous isoprene, and that isoprene-emitting plants that are sensitive to ozone do not suffer damage when exposed to ozone. Isoprene-induced ozone resistance is associated with the maintenance of photochemical efficiency and with a low energy dissipation, as indicated by fluorescence quenching. This suggests that isoprene effectively stabilizes thylakoid membranes. However, when isoprene reacts with ozone within the leaves or in a humid atmosphere, it quenches the ozone concentration to levels that are less or non-toxic for plants. Thus, protection from ozone in plants fumigated with isoprene may be due to a direct ozone quenching rather than to an induced resistance at membrane level. Irrespective of the mechanism, isoprene is one of the most effective antioxidants in plants.     

FTIR microspectroscopy study of composition fluctuations in extruded amylopectin-gelatin blends

The spatial variation in the composition of nonexpanded biopolymer blends prepared by extrusion of mixtures of gelatin with either native or pregelatinized waxy maize starch was studied using a 30-microm aperture FTIR microspectroscopy technique. The ratio of the areas of the "saccharide" bands (953-1180 cm(-1)) and the amide I and II bands (1483-1750 cm(-1)) was used to monitor the relative distributions of the two components of the blend. Two calibration methods were used to obtain amylopectin concentration values from the ratios of the IR bands. The results suggested a high degree of heterogeneity in these blends, despite the thorough mixing expected by twin-screw extrusion processing. The concentration fluctuations were greater for the blends produced by extruding gelatin and native waxy maize starch mixtures. This was in agreement with the reduced degree of conversion of the starch granules when extruded in the presence of gelatin. The FTIR 2-dimensional maps obtained suggested that in the blends produced from either native or pregelatinized starch at all concentrations studied (25/75, 50/50, and 75/25 amylopectin/gelatin) the gelatin constituted the continuous phase. The effect of the spatial resolution on the FTIR microspectroscopy results was considered and the proposed interpretation was verified by the use of polarized light microscopy and FTIR microspectroscopy acquired at higher spatial resolution (10 microm).     

Effect of spermine on the efficiency and fidelity of the codon-specific binding of tRNA to the ribosomes

Binding of the yeast Tyr-tRNA and Phe-tRNA to the A site, and the binding of their acetyl derivatives to the P site of poly(U11,A)-programmed Escherichia coli ribosomes was studied. Spermine stimulated the rate of binding of both tRNAs at least threefold, enabling more than 90% final saturation of both ribosomal binding sites. The effect is observed when the tRNAs, but not ribosomes or poly(U11,A), are preincubated with polyamine. Regardless of the binding site, optimal saturation was reached at spermine/tRNA molar ratios of 3 for tRNA(Phe) and 5 for tRNA(Tyr). The same low spermine/tRNA ratios were previously reported to stabilize the conformation of these tRNAs in solution. On the other hand, the messenger-free, EF-Tu- and EF-G-dependent polymerization of lysine from E. coli Lys-tRNA is drastically reduced, while the poly(A)-directed polymerization is stimulated by spermine through a wide range of Mg2+ concentrations. Misreading of UUU codons as isoleucine, assayed by the A-site binding of E. coli Ile-tRNA, is also inhibited by spermine. All these results demonstrate that spermine increases the efficiency and accuracy of a series of macromolecular interactions leading to the correct incorporation of an amino acid into protein, at the same time preventing some unspecific or erroneous interactions. From the analogy with its known structural effects, it can be inferred that spermine does so by conferring on the tRNA a specific biologically functional conformation.