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Decreasing progressive dermal ischemia after burning could theoretically limit the amount of skin necrosis. It is controversial whether the use of free radical scavengers could prevent the progressive dermal ischemia of the postburn stasis zone. We evaluated the effect of superoxide dismutase (SOD) on preventing postburn dermal ischemia in animal models by the India ink perfusion and skin transparent preparation techniques. The closely clipped backs of guinea-pigs were bathed in 75 degrees C water for 10 s. Within 5 min postburn, SOD-treated groups were administered SOD (10,000 u/kg) intra-peritoneally every 6 h. All animals were perfused with 70 per cent India ink via cervical artery cannula by 16 kPa constant pressure at 0, 8, 16, 24 h postburn, and the skin transparent preparations were made, while the level of malonyl dialdehyde (MDA) in skin tissue was assessed. The results showed that with prolongation of postburn time, the rate of filling of India ink in skin capillary plexuses decreased gradually (p < 0.01). MDA increased continuously, which was related to postburn dermal ischemia (r = 0.689, p < 0.01). Though the level of MDA decreased in SOD-treated groups, the India ink filling rates showed no significant difference between controls and experimental groups (p > 0.05). The results were also confirmed by observation of skin transparent preparations and TEM. This study suggests that superoxide dismutase fails to prevent progressive dermal ischemia after burning.
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PMID:Can superoxide dismutase prevent postburn dermal ischemia? 923 83

Transmission of Pseudodiplorchis americanus is restricted to the brief period when its host, a desert toad, enters water to spawn. The parasite accumulates its entire annual reproductive output within an elongated uterus during the 10-11-month period of host hibernation. Embryos of P. americanus, at all stages of development, are retained within the uterus which eventually becomes packed with around 150 encapsulated infective larvae. Recently formed eggs, which comprise a fertilized ovum and 2-3 vitelline cells, are closely surrounded by a primary eggshell which stains positively for acidic proteins and keratin. Initially, during passage along the proximal uterus, the egg capsule is only 60 microns in diameter, but as it passes to the distal uterus it expands to 800 microns in diameter to accommodate the growing larva. Due to chemical alterations or complete replacement of the shell, the final (secondary) egg capsule is a large sac-like structure composed of elastin. The flexible nature of this shell maximizes the numbers of infective larvae which can be stored in utero. TEM studies have revealed this capsule to be composed of multi-laminate membranes with a specialized cytoplasmic lining involved in a unique mechanism for embryo nutrition. This is the first report of an elastin-type eggshell within the Monogenea.
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PMID:Histological analysis of the egg capsule of the ovoviviparous polystomatid monogenean, Pseudodiplorchis americanus. 936 90

Gluma Dentin Bond is an adhesive system, where the primer contains 5% glutaraldehyde and 35% hydroxyethyl methacrylate. Practitioners have reported a strong desensitizing effect of the Gluma system on dentin. This study, thus, sought to evaluate the effect of this system on dentin using various microscopic techniques. 12 non-restored human molars extracted for prosthodontic reasons were used. Prior to extraction the buccal cusps were removed such that a 2 mm x 2 mm wide dentin surface was exposed. The surfaces were treated in 6 ways: (1) application of Gluma 2 cleanser, Gluma 3 primer to which 0.1% w/v fluorescein was added, and Gluma 4 sealer; (2) as in (1) but treatment with H2O/0.1% w/v fluorescein instead of the Gluma 3; (3) as in (1) but without Gluma 2; (4) as in (1) but with application of 5% glutaraldehyde instead of Gluma 3; (5) as in (1) but without Gluma 4; (6) as in (1) but with application of 35% HEMA/0.1% w/v fluorescein instead of Gluma 3. Following extraction, 1 tooth per procedure was prepared for confocal laser scanning microscopy. The remaining teeth were fixed and prepared for SEM and TEM evaluation. In specimens of procedures (1) and (5), tubular occlusions could be seen to a depth of 200 microm. In specimens of procedure (4) tubular occlusions were found only to a depth of 50 microm. Such occlusions were not seen in control specimens (2), in specimens where the smear-layer had not been removed (3), or following application of HEMA alone (6). It is concluded that glutaraldehyde can intrinsically block dentinal tubules. The septa in the tubules may counteract the hydrodynamic mechanism for dentinal sensitivity.
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PMID:Closing of dentinal tubules by Gluma desensitizer. 939 2

It is well known that arginine vasopressin (AVP) produces up to a 40-fold increase (0.1 to 4.0 microL/min.cm2) in net water flux across the amphibian urinary bladder under an osmotic gradient (mucosal side 10% hypotonic). No AVP effect is observed when the gradient is in the opposite direction (serosal hypotonic). Similar asymmetrical behavior to osmotic gradients occurs in the frog corneal epithelium. This rectification phenomenon has not been satisfactorily explained. We measured net water fluxes in bladder sacs and confirmed that AVP has no effect when the serosal bath is hypotonic. We reasoned that the 'abnormal' serosal osmolarity was inducing changes in membrane water permeability, the very parameter being measured. Thus, we studied the effect of solution osmolarity on diffusional water flow (Jdw) across the frog bladder using 3H2O. As expected, AVP doubled Jdw (in either direction from 12 to 21 microL/min.cm2) when the serosal solution was iso-osmolar regardless of mucosal osmolarity. However, in the AVP-stimulated bladders, hypo-osmolarity of the serosal solution reduced Jdw by 42%, an effect that was reversible when normal osmolarity was re-established. Amphotericin B (instead of AVP) was used to irreversibly increase the permeability to water of the apical membrane. Under these conditions, basolateral hypotonicity also reversibly decreased Jdw by 32%, suggesting the basolateral membrane as the site where permeability is reduced. SEM and TEM of the tissue shows extreme swelling when it was exposed to serosal hypotonicity with or without AVP and typical surface morphology changes following hormone stimulation. We conclude that this swelling may initiate a signaling mechanism that reduces basolateral water permeability. These findings constitute evidence of basolateral water channel permeability regulation, which can also contribute to cell volume regulation.
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PMID:Evidence of basolateral water permeability regulation in amphibian urinary bladder. 946 4

The crystal structure of a phosphonate complex of the class A TEM-1 beta-lactamase has been determined to a resolution of 2.0 A. The phosphonate appears stoichiometrically at the active site, bound covalently to Ser70Ogamma, with one phosphonyl oxygen in the oxyanion hole. Although the overall structure is very similar to that of the native enzyme (rms difference 0.37 A for all heavy atoms), changes have occurred in the position of active site functional groups. The active site is also not in the conformation observed in the complex of another class A beta-lactamase, that of Staphylococcus aureus PC1, with the same phosphonate [Chen, C. C. H., et al. (1993) J. Mol. Biol. 234,165-178]. Both phosphonate structures, however, can be seen to represent models of acylation transition-states since in each the deacylating water molecule appears firmly bound to the Glu166 carboxylate group. The major difference between the structures lies in the positioning of Lys73Nzeta and Ser130Ogamma. In the S. aureus structure, the closest interaction of these functional groups is between Lys73Nzeta and Ser70Ogamma (2.8 A), while in the TEM-1 structure it is between Ser130Ogamma and the second phosphonyl oxygen of the bound inhibitor (2.8 A). The former structure therefore may resemble a transition state for formation of the tetrahedral species in acylation by nucleophilic attack on the substrate, where Lys73Nzeta presumably catalyzes the reaction as a general base. The TEM-1 structure can then be seen as an analogue of the transition state for breakdown of the tetrahedral species, where Ser130Ogamma is acting as a general acid, assisting the departure of the leaving group. The class A beta-lactamase crystal structures now available lead to a self-consistent proposal for a mechanism of catalysis by these enzymes.
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PMID:Crystal structure of an acylation transition-state analog of the TEM-1 beta-lactamase. Mechanistic implications for class A beta-lactamases. 948 12

A procedure is presented for the preparation of thin layers of vitrified biological suspensions in the presence of ammonium molybdate, which we term cryo-negative staining. The direct blotting of sample plus stain solution on holey carbon supports produces thin aqueous films across the holes, which are routinely thinner than the aqueous film produced by conventional negative staining on a continuous carbon layer. Because of this, a higher than usual concentration of negative stain (ca. 16% rather than 2%) is required for cryo-negative staining in order to produce an optimal image contrast. The maintenance of the hydrated state, the absence of adsorption to a carbon film and associated sample flattening, together with reduced stain granularity, generates high contrast cryo-images of superior quality to conventional air-dry negative staining. Image features characteristic of unstained vitrified cryo-electron microscopic specimens are present, but with reverse contrast. Examples of cryo-negative staining of several particulate biological samples are shown, including bacteriophage T2, tobacco mosaic virus (TMV), bovine liver catalase crystals, tomato bushy stunt virus (TBSV), turnip yellow mosaic virus (TYMV), keyhole limpet hemocyanin (KLH) types 1 and 2, the 20S proteasome from moss and the E. coli chaperone GroEL. Densitometric quantitation of the mass-density of cryo-negatively stained bacteriophage T2 specimens before and after freeze-drying within the TEM indicates a water content of 30% in the vitreous specimen. Determination of the image resolution from cryo-negatively stained TMV rods and catalase crystals shows the presence of optical diffraction data to ca. 10 A and 11.5 A, respectively. For cryo-negatively stained vitrified catalase crystals, electron diffraction shows that atomic resolution is preserved (to better than 20 diffraction orders and less than 3 A). The electron diffraction resolution is reduced to ca. 10 A when catalase crystal specimens are prepared without freezing or when they are freeze-dried in the electron microscope. Thin vitrified films of TMV, TBSV and TYMV in the presence of 16% ammonium molybdate show a clear indication of two-dimensional (2-D) order, confirmed by single particle orientational analysis of TBSV and 2-D crystallographic analysis of TYMV. These observations are in accord with earlier claims that ammonium molybdate induces 2-D array and crystal formation from viruses and macromolecules during drying onto mica. Three-dimensional analysis of the TBSV sample using the tools of icosahedral reconstruction revealed that a significant fraction of the particles were distorted. A reconstruction from a subset of undistorted particles produced the characteristic T = 3 dimer clustered structure of TBSV, although the spikes are shortened relative to the structure defined by X-ray crystallography. The 20S proteasome, GroEL, catalase, bacteriophage T2, TMV, TBSV and TYMV all show no indication of sample instability during cryo-negative staining. However, detectable dissociation of the KLH2 oligomers in the presence of the high concentration of ammonium molybdate conforms with existing knowledge on the molybdate-induced dissociation of this molecule. This indicates that the possibility of sample-stain interaction in solution, prior to vitrification, must always be carefully assessed.
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PMID:Cryo-negative staining. 968 50

Phospholipid hydrolysis to free fatty acid and 1-lyso-phospholipid by water-soluble phospholipase A2 (PLA2) at the surface of lipid membranes exhibits a poorly understood transition from a low-activity lag phase to a burst regime of rapid hydrolysis. Understanding this kinetic phenomenon may increase our insight into the function of PLA2 under physiological conditions as well as into general interfacial catalysis. In the present study we apply for the first time cryo-transmission electron microscopy (cryo-TEM) and high-performance liquid chromatography (HPLC) to characterize the PLA2 hydrolysis of phospholipid vesicles with respect to changes in lipid composition and morphology. Our direct experimental results show that the initial reaction conditions are strongly perturbed during the course of hydrolysis. Most strikingly, cryo-TEM reveals that starting in the lag phase, vesicles become perforated and degrade into open vesicles, bilayer fragments, and micelles. This structural instability extends throughout the system in the activity burst regime. In agreement with earlier reported correlations between initial phospholipase activity and substrate morphology, our results suggest that the lag-burst phenomenon reflects a cascade process. The PLA2-induced changes in lipid composition transform the morphology which in turn results in an acceleration of the rate of hydrolysis because of a strong coupling between the PLA2 activity and the morphology of the lipid suspension.
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PMID:Direct imaging by cryo-TEM shows membrane break-up by phospholipase A2 enzymatic activity. 969 92

In order to examine the effects of alcohol on the hard palatine mucosa of rats, sixty adult female rats (Rattus norvegicus albinus) were divided into two experimental groups. The control group received solid diet (Purina rat chow) and tap water ad libitum. The alcoholic group received the same solid diet and was allowed to drink only sugar cane brandy dissolved in 30% Gay Lussac (v/v). At the end of periods of 90, 180 and 270 days of treatment, the animals at estro were sacrificed and the hard palatine mucosa were prepared for TEM and SEM methods. The basal cells of the alcoholic groups (90, 180 and 270 days of treatment) demonstrated some alterations: the intercellular spaces between these cells were higher, presented cytoplasmatic lipid droplets and autolysis. Also, the connective tissue showed intense lipid droplets accumulation in the alcoholic groups. These modifications suggested that chronic alcohol ingestion was able to modify the integrity of the cells in the rat hard palatine mucosa.
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PMID:Morphological changes on the hard palatine mucosa of rats (Rattus norvegicus albinus) after chronic alcohol consumption. 972 98

A prepration technique for nanometer particles, namely the replacing solvent drying technique, was developed. The main process of the technique including replacement of water in gel with special organic solvent and the removal of the solvent by distillation. No collapse of gel structure took place because of low interface tension between water and the solvent as well as low surface tension of the solvent. Where special apparatus and strictly limited preparation conditions were not necessary. The technique is noted for its low preparation cost and high universality. Titanium oxide and copper borate were prepared using the technique and were characterized using XRD, nitrogen physical adsorption, TEM, and small angle X-ray scattering. Results indicated that the titanium oxide and copper borate possessed particle sizes of 7-10 and 7-20 nm as well as a specific surface area of 235 m2/g and 360 m2/g, respectively. The specific surface area were even much higher than that of the samples prepared using supercritical drying technique. Copyright 1998 Academic Press.
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PMID:Replacing Solvent Drying Technique for Nanometer Particle Preparation. 984 79

The aggregation behavior of Tyloxapol, a nonionic surfactant oligomer with a repeating unit close to Triton X-100 (TX100), and a maximum degree of polymerization of about 7, has been investigated in aqueous solution by means of fluorescence probing, time-resolved fluorescence quenching (TRFQ) and transmission electron microscopy at cryogenic temperature (cryo-TEM). The plot of the pyrene fluorescence intensity ratio I1/I3 against the Tyloxapol concentration shows no clear evidence of a critical micelle concentration contrary to TX100. Nevertheless, the fitting of these data, assuming a partition of pyrene between Tyloxapol aggregates and water, yields cmc values in the micromolar range, i.e., about a hundred times lower than for the "monomer" TX100. The values of I1/I3 at high surfactant concentrations indicate that Tyloxapol micelles provide pyrene a less polar environment than TX100 micelles. The use of the viscosity-sensitive probe 1,3-dipyrenylpropane indicates that the microviscosity of Tyloxapol micelles is quite high, three to four times larger than that for TX100 micelles, and decreases rapidly with increasing temperature. Also the microviscosities of both TX100 and Tyloxapol micelles are larger than those for the micelles of the nonionic ethoxylated surfactant C12E9. The aggregation numbers of Tyloxapol and of TX100 micelles measured using TRFQ increase with temperature, with the Tyloxapol micelles being smaller than the TX100 micelles. Cryo-TEM shows that the Tyloxapol micelles remain spheroidal up to a concentration of about 10 wt%. At 15 wt%, some regions of ordered elongated micelles are also observed which may be the precursors of the hexagonal phase known to occur at about 35 wt%. Copyright 1999 Academic Press.
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PMID:Aggregation Behavior of Tyloxapol, a Nonionic Surfactant Oligomer, in Aqueous Solution. 992 3


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