Cryoelectron microscopy of a nucleating model bile in vitreous ice: formation of primordial vesicles.
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Because gallstones form so frequently in human bile, pathophysiologically relevant supersaturated model biles are commonly employed to study cholesterol crystal formation. We used cryo-transmission electron microscopy, complemented by polarizing light microscopy, to investigate early stages of cholesterol nucleation in model bile. In the system studied, the proposed microscopic sequence involves the evolution of small unilamellar to multilamellar vesicles to lamellar liquid crystals and finally to cholesterol crystals. Small aliquots of a concentrated (total lipid concentration = 29.2 g/dl) model bile containing 8.5% cholesterol, 22.9% egg yolk lecithin, and 68.6% taurocholate (all mole %) were vitrified at 2 min to 20 days after fourfold dilution to induce supersaturation. Mixed micelles together with a category of vesicles denoted primordial, small unilamellar vesicles of two distinct morphologies (sphere/ellipsoid and cylinder/arachoid), large unilamellar vesicles, multilamellar vesicles, and cholesterol monohydrate crystals were imaged. No evidence of aggregation/fusion of small unilamellar vesicles to form multilamellar vesicles was detected. Low numbers of multilamellar vesicles were present, some of which were sufficiently large to be identified as liquid crystals by polarizing light microscopy. Dimensions, surface areas, and volumes of spherical/ellipsoidal and cylindrical/arachoidal vesicles were quantified. Early stages in the separation of vesicles from micelles, referred to as primordial vesicles, were imaged 23-31 min after dilution. Observed structures such as enlarged micelles in primordial vesicle interiors, segments of bilayer, and faceted edges at primordial vesicle peripheries are probably early stages of small unilamellar vesicle assembly. A decrease in the mean surface area of spherical/ellipsoidal vesicles was correlated with the increased production of cholesterol crystals at 10-20 days after supersaturation by dilution, supporting the role of small unilamellar vesicles as key players in cholesterol nucleation and as cholesterol donors to crystals. This is the first visualization of an intermediate structure that has been temporally linked to the development of small unilamellar vesicles in the separation of vesicles from micelles in a model bile and suggests a time-resolved system for further investigation. (+info)
Cholesteryl ester hydrolysis in J774 macrophages occurs in the cytoplasm and lysosomes.
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The relationship of cholesteryl ester hydrolysis to the physical state of the cholesteryl ester in J774 murine macrophages was explored in cells induced to store cholesteryl esters either in anisotropic (ordered) inclusions or isotropic (liquid) inclusions. In contrast to other cell systems, the rate of cholesteryl ester hydrolysis was faster in cells containing anisotropic inclusions than in cells containing isotropic inclusions. Two contributing factors were identified. Kinetic analyses of the rates of hydrolysis are consistent with a substrate competition by co-deposited triglyceride in cells with isotropic inclusions. In addition, hydrolysis of cholesteryl esters in cells with anisotropic droplets is mediated by both cytoplasmic and lysosomal lipolytic enzymes, as shown by using the lysosomotropic agent, chloroquine, and an inhibitor of neutral cholesteryl ester hydrolase, umbelliferyl diethylphosphate. In cells containing anisotropic inclusions, hydrolysis was partially inhibited by incubation in media containing either chloroquine or umbelliferyl diethylphosphate. Together, chloroquine and umbelliferyl diethylphosphate completely inhibited hydrolysis. However, when cells containing isotropic inclusions were incubated with umbelliferyl diethylphosphate, cholesteryl ester hydrolysis was completely inhibited, but chloroquine had no effect. Transmission electron microscopy demonstrated a primarily lysosomal location for lipid droplets in cells with anisotropic droplets and both non-lysosomal and lysosomal populations of lipid droplets in cells with isotropic droplets. These results support the conclusion that there is a lysosomal component to the hydrolysis of stored cholesteryl esters in foam cells. (+info)
Most calcium pyrophosphate crystals appear as non-birefringent.
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OBJECTIVE: To determine the proportion of calcium pyrophosphate dihydrate (CPPD) crystals that appear as non-birefringent when observed under the polarised light microscope. METHODS: Two observers examined independently 10 synovial fluid samples obtained during an episode of arthritis attributable to CPPD crystals. Ten synovial fluid samples from patients with acute gout were used as a reference. The examination was performed after placing a fluid sample in a Niebauer haemocytometric chamber; a crystal count was done first under ordinary light, then in the area corresponding to a 0.1 ml, under polarised light RESULTS: The percentages of birefringence appreciated for CPPD were 18% (confidence intervals (CI) 12, 24) for observer 1, and 17% (CI 10, 24) for observer 2 (difference NS). The percentages of birefringence for monosodium urate were 127% (CI 103, 151) for observer 1 and 107% (CI 100, 114) for observer 2 (difference NS). Percentages above 100% indicate that crystals missed under ordinary light became apparent under polarised light. CONCLUSION: Only about one fifth of all CPPD crystals identified by bright field microscopy show birefringence when the same synovial fluid sample is observed under polarised light. If a search for CPPD crystals is conducted under polarised light, the majority of the crystals will be missed. Ordinary light allows a better rate of CPPD crystal detection but observation under polarised light of crystals showing birefringence is required for definitive CPPD crystal identification. (+info)
An ethogram of body patterning behavior in the biomedically and commercially valuable squid Loligo pealei off Cape Cod, Massachusetts.
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Squids have a wide repertoire of body patterns; these patterns contain visual signals assembled from a highly diverse inventory of chromatic, postural, and locomotor components. The chromatic components reflect the activity of dermal chromatophore organs that, like the postural and locomotor muscles, are controlled directly from the central nervous system. Because a thorough knowledge of body patterns is fundamental to an understanding of squid behavior, we have compiled and described an ethogram (a catalog of body patterns and associated behaviors) for Loligo pealei. Observations of this species were made over a period of three years (> or = 440 h) and under a variety of behavioral circumstances. The natural behavior of the squid was filmed on spawning grounds off Cape Cod (northwestern Atlantic), and behavioral trials in the laboratory were run in large tanks. The body pattern components--34 chromatic (including 4 polarization components), 5 postural, and 12 locomotor--are each described in detail. Eleven of the most common body patterns are also described. Four of them are chronic, or long-lasting, patterns for crypsis; an example is Banded Bottom Sitting, which produces disruptive coloration against the substrate. The remaining seven patterns are acute; they are mostly used in intraspecific communication among spawning squids. Two of these acute patterns--Lateral Display and Mate Guarding Pattern--are used during agonistic bouts and mate guarding; they are visually bright and conspicuous, which may subject the squids to predation; but we hypothesize that schooling and diurnal activity may offset the disadvantage presented by increased visibility to predators. The rapid changeability and the diversity of body patterns used for crypsis and communication are discussed in the context of the behavioral ecology of this species. (+info)
New microscope gives scientists the inside scoop on living cells.
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Scientists at Pacific Northwest National Laboratory in Richland, Washington, have developed a unique imaging system capable of focusing on a single living cell within an organism. This new technology will be used in what the multidisciplinary team has termed a "cellular observatory" to study the effect of environmental insults to live cells. (+info)
Mechanical behaviour of plant tissues: composite materials or structures?
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The mechanical characteristics of the strengthening tissue of young axes of Aristolochia macrophylla were studied in successive loading-unloading cycles in tension. Elastic, viscoelastic and plastic deformations could be distinguished. After the first cycle, the material was in a state different from its original state, to which it returned only partially and/or slowly. Internal 'microstructural' prestresses are considered as an explanation for the mechanical behaviour seen in Aristolochia macrophylla and several other plants. (+info)
Oxalate-producing pulmonary aspergillosis in an alpaca.
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An aging lactating alpaca was presented in sternal recumbency. Although bright and alert, she did not respond to symptomatic treatment and was euthanized 5 weeks after initial presentation. Gross postmortem examination revealed purulent material in the pulmonary airways. Histologic examination of the lungs revealed an extensive pyogranulomatous pneumonia with bronchiectasis. There were abundant fungal hyphae and high numbers of associated oxalate crystals, which were presumed to have been produced by the fungus. Low numbers of yeast cells were also present. Microbiological culture of tissues on horse blood agar and Sabouraud's agar identified the fungus to be Aspergillus niger. There was also moderate growth of Candida albicans. Calcium oxalate crystals in cytologic and histologic preparations can suggest an underlying Aspergillus infection. This is the first reported veterinary case of pneumonia due to Aspergillus niger infection and the associated production of oxalate crystals. (+info)
Transregulation of adenylyl-cyclase-coupled inhibitory receptors in heart failure enhances anti-adrenergic effects on adult rat cardiomyocytes.
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OBJECTIVE: In congestive heart failure (CHF), a desensitisation of stimulatory beta-receptors and of adenylyl cyclase in the heart is associated with an increase in inhibitory Gi proteins. To investigate whether the regulation of the Gi-mediated inhibitory side of the adenylyl cyclase system may be of functional importance in the failing myocardium, the contractile response of isolated adult cardiomyocytes to stimulation of inhibitory muscarinic M2 and A1 adenosine receptors was analysed. METHODS: CHF was induced in rats by banding of the ascending aorta and was verified by doubling of lung wet weight. After four weeks, contraction amplitude (delta L) and the velocity (dL/dtmax) of isolated ventricular cardiomyocytes during electrical field stimulation in the presence of 1 mM Ca2+ were measured using video micrometry. RESULTS: Contractile responses of failing cardiomyocytes to 5 mM Ca2+ were unchanged. The response to increasing concentrations of the beta-adrenergic agonist, isoproterenol (0.1-30 nM), and to forskolin (0.1 nM-1 microM) were significantly blunted. When A1 receptors were activated with N6-(R-phenyl-isopropyl)-adenosine (PIA; 0.01-1 microM) in the presence of 3 nM isoproterenol, contractility was unchanged in cells compared with those from sham-operated rats, but delta L was reduced by up to 23% and dL/dtmax by 35% in failing cardiomyocytes (P < 0.01), demonstrating an enhanced inhibitory effect of A1 receptors. The response to the M2 receptor agonist, carbachol (0.01-3 microM), was augmented to a comparable extent (delta L, -22%, dL/dtmax, -39%; P < 0.01). CONCLUSIONS: In CHF, the inotropic responses to beta-receptor-stimulation and to direct stimulation of adenylyl cyclase, but not to Ca2+, are diminished due to desensitisation of the stimulatory side of the adenylyl cyclase signal transduction system. In parallel, the responses to inhibitory receptors are augmented, leading to a pronounced Gi-mediated negative inotropic effect on failing heart muscle cells. Those anti-adrenergic effects could contribute to the contractile dysfunction of the failing heart. Reversal of the sensitisation to inhibitory stimuli might be one of the desirable mechanisms of medical therapy in CHF. (+info)