Biomedical imaging: 2001 and beyond.
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Anatomy↗
Biomedical subjects
D S Lester
Publications and source records attributed to D S Lester.
Redefining rats, mice, and birds. AAA (American Association of Anatomists) Public Affairs Committee.
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Animal Rights↗
3-Dimensional visualization of lesions in rat brain using magnetic resonance imaging microscopy.
High-resolution (< 50 microm) magnetic resonance imaging microscopy (MRM) has been used to identify brain regions and localization of excitotoxin-induced lesions in fixed rat brains, subsequently confirmed using standard histology. The anatomical extent of lesions identified by MRM was identical to that seen in histological sections and various histopathological changes could be visualized. In contrast to the time involved in preparing and examining histological sections, lesions in intact brains could be rapidly identified and visualized in three dimensions by examining digitally generated sections in any plane. This study shows that MRM has tremendous potential as a prescreening tool for neurotoxicity and neuropathology. These observations suggest that MRM has the potential to affect pathology much as conventional MRI has influenced clinical imaging.
Animals↗
Infrared microspectroscopic imaging of the cerebellum of normal and cytarabine treated rats.
Conventionally, the diagnosis of neuropathology in a subject requires the identification of a behavioral modification, which provides direction for appropriate histological analyses. However, since the ultimate diagnosis of the pathology largely depends on the initial choice of histological tests, the opportunity exists for inaccurate or insensitive results. An innovative approach using Fourier transform infrared (FT-IR) spectroscopic imaging to diagnose neuropathology should prove useful. This novel method monitors and visualizes the underlying chemistry of the tissue, based on hundreds of vibrational absorption bands that are intrinsic to the sample. As such, it makes no prior assumptions as to the type or degree of pathology. Using this technique, we have spectroscopically imaged cerebellar tissue slices from rats [control subjects and subjects treated with the antineoplastic drug, cytarabine (Ara-C)], and have been able to correlate lipid and protein distributions within distinct cell types in the cerebellum. A further benefit of the technique is that it simultaneously records tens of thousands of independent spectra from different spatial locations within the sample. Thus, a variety of statistical and multivariate techniques can be exploited to characterize large sample areas and to provide robust classification of individual spectral signatures. In comparison to standard histological protocols, FT-IR spectroscopic imaging simultaneously analyzes cell layers and identifies subtle structural and biochemical changes within the sample. We suggest that FT-IR spectroscopic imaging should provide a highly reliable, complementary tool for standard histological tier testing.
Animals↗
Time-resolved imaging of protein kinase C activation during sea urchin egg fertilization.
To study protein kinase C (PKC) activation during sea urchin egg fertilization we used three different fluorescent probes specific for PKC, namely, fim-1, which recognizes the catalytic site of the enzyme, and BODIPY- and NBD-phorbol esters interacting with the PKC regulatory domain. We were able to follow PKC activation during the early steps of fertilization, the three different probes giving the same fluorescent pattern. Within 120 s following insemination, the fluorescent signal increased and clustered in the cortical zone of the cell. The process was Ca2+ dependent and was inhibited in the presence of staurosporine, a PKC inhibitor. According to our in vitro probe characterization, this signal increase is due to PKC activation. These findings were further confirmed by Western blot analysis. This initial phase was followed by a rapid decrease which might be attributed to PKC hydrolysis by Ca2(+)-dependent proteases. The kinetics and the site distribution of PKC activation appear in complete agreement with the putative functions previously suggested for PKC during fertilization.
Animals↗
Applications of fourier transform infrared imaging microscopy in neurotoxicity.
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Nervous System↗
Synergistic activation of protein kinase C by arachidonic acid and diacylglycerols in vitro: generation of a stable membrane-bound, cofactor-independent state of protein kinase C activity.
The present study examines the synergistic activation of PKC by arachidonic acid and diacylglycerols in phospholipid vesicles and demonstrates that this combination of activators leads to the formation of a constitutively active, phospholipid-bound form of the enzyme. Activation of PKC was almost entirely calcium-dependent with vesicles containing dioleoylglycerol alone. In contrast, considerable calcium-independent activity was observed when vesicles contained both a diacylglycerol and free arachidonic acid. High-affinity association of enzyme activity with diacylglycerol-containing vesicles was calcium dependent and reversible. However, addition of arachidonic acid to diacylglycerol-containing vesicles resulted in irreversible PKC binding in the absence of calcium. Immunoblot analysis indicated that the calcium-independent binding was not isozyme-specific. The activity of the vesicle-associated PKC, bound to vesicles in the absence of calcium, was predominantly calcium-dependent. On the other hand, when the binding and isolation of vesicle-bound enzyme was conducted in the presence of calcium, the subsequent activity was almost entirely resistant to calcium chelation. This vesicle-associated form of the enzyme, when detergent extracted and recombined with phospholipid vesicles, maintained significant 'constitutive' activity (activity in the absence of both diacylglycerol and calcium). The data from this in vitro system provide the basis for a model of the physiological regulation of PKC in which the combined actions of arachidonate and diacylglycerol facilitate the stable formation of a tightly membrane-associated, intrinsically active form of PKC.
Animals↗
High-resolution fluorescent labeling of living cerebellar slices.
In the present study, we extend previous research on staining of living brain slices with fluorescent phospholipids. This new procedure allows high-resolution staining of specific cell types, in particular, Purkinje cells, in the cerebellar slice while not affecting the intrinsic electrical activity of the tissue. Four different nitrobenzoxadiole (NBD)-phospholipids were incorporated into living cerebellar slices via loading from small unilamellar vesicles (SUVs), composed of a carrier and the fluorescent lipid. The labeled acidic phospholipid, NBD-phosphatidic acid (NBD-PA), produced the highest resolution images with exquisite labeling of the dendritic fields. The label was incorporated predominantly into the Purkinje cell body (excluding the nucleus), with more diffuse staining in other cell types, including stellate, basket and granule cells. The labeled lipid concentration and composition of the carrier lipid were significant in determining the specificity of labeling. Labeling, which was optimal after a 1 h incubation, was present throughout the depth of the slice. This procedure provides a promising approach to fluorescent labeling that will allow simultaneous monitoring of changes in cellular morphology and electrophysiology of living brain slices.
4-Chloro-7-nitrobenzofurazan↗
Human olfactory neuroepithelial cells: tyrosine phosphorylation and process extension are increased by the combination of IL-1beta, IL-6, NGF, and bFGF.
Olfactory neuroepithelial cells (ONC) grown from biopsies of human donors are a novel cell culture system that may facilitate studies into normal and disease-related human neurobiology. We further characterized the expression of cell surface markers and intermediate filaments, and responses to neurotrophic factors by ONC. ONC are positive for cell surface markers N-CAM, PSA-N-CAM, neutral endopeptidase, N-aminopeptidase, NGF low-affinity receptor homologue (CD40), and transferrin receptor by flow cytometry for the intermediate filament proteins peripherin, vimentin, and NF-H by immunocytochemistry. Responses to neurotrophic factors measured were process outgrowth, cytoskeletal protein expression, and protein phosphorylation. Process outgrowth was increased by interleukin-beta 164-171 (IL-1beta) or by the combination of IL-1beta, interleukin-6 (IL-6), nerve growth factor (NGF), and basic fibroblast growth factor (bFGF). This combination of IL-1beta, IL-6, NGF, and bFGF (16NF) increased expression of two cytoskeletal proteins, NF-H protein and microtubule-associated protein tau. Application of the individual neurotrophic factors IL-1beta, IL-6, NGF, and bFGF increased protein phosphorylation, while 16NF produced an immediate increase in tyrosine phosphorylation of several proteins (MW of 40-80, 120, 150, and 190 kDa). The 16NF combination appears to act through a tyrosine-kinase-mediated pathway to induce process extension and increase NF-H expression. The ONC culture has the potential to be further explored to examine the relationship among process outgrowth, protein phosphorylation, and synergy between neurotrophin and cytokine receptor systems.
Antigens, CD↗
Translocation and activation of protein kinase C by the plasma cell tumor-promoting alkane pristane.
Pristane (2,6,10,14-tetramethylpentadecane) is a C19-isoalkane that promotes the development of plasmacytomas in genetically susceptible BALB/c mice. Similarities between the effects of pristane and protein kinase C (PKC)-activating phorbol esters suggested that the tumor promoting activity of pristane might involve the activation of PKC. Here we show that up to 5 mol% of pristane can be homogeneously incorporated into phosphatidylcholine/phosphatidylserine bilayers. Membrane-incorporated pristane partially activated PKC and increased phorbol ester binding to the bilayer by more than 50%. Pristane (50 microM) delivered as an inclusion complex with beta-cyclodextrin to promyelocytic HL-60 leukemia cells induced a partial long-term translocation of PKC to the cell membrane. This was accompanied by differentiation of HL-60 cells into macrophage-like cells. It is concluded that activation of PKC may comprise an important aspect of the tumor promoting potential of pristane.
Animals↗
Imaging protein kinase C activation in living sea urchin eggs after fertilization.
The fluorescent dye NBD-phorbol acetate was used to visualize the activation of protein kinase C (PKC) in living Lytechinus pictus eggs during fertilization. The dye interacts directly with PKC as determined using a competitive binding assay. Quantitative image analysis of sequential images from laser-scanning confocal microscopy showed a significant reorganization of the signal in the vicinity of the cortical granules and the plasma membrane that began immediately following fertilization and persisted up to 1 hr (P<0.0001). At the concentrations employed, the NBD-phorbol dye was not capable of inducing a significant translocation of the fluorescent signal to the membrane, nor did it appear to interfere with the cell cycle. It therefore seems likely that the present in vivo results reflect the previously reported in vitro activation of protein kinase C immediately subsequent to fertilization. Such an interpretation is parsimonious with the results of parallel subcellular fractionation experiments using an N-terminal polyclonal antibody to sea urchin PKC which showed a significant (P<0.037) translocation of the enzyme from the cytosolic fraction to the membrane fraction 40 min subsequent to fertilization. This study supports and extends previous in vitro data suggesting that PKC activation subsequent to fertilization occurs at or near the egg plasma membrane, perhaps in association with arachadonic acid-rich cortical granules.
Animals↗
Effects of diacylglycerols on conformation of phosphatidylcholine headgroups in phosphatidylcholine/phosphatidylserine bilayers.
The effects of five diacylglycerols (DAGs), diolein, 1-stearoyl,2-arachidonoyl-sn-glycerol, dioctanoylglycerol, 1-oleoyl,2-sn-acetylglycerol, and dipalmitin (DP), on the structure of lipid bilayers composed of mixtures of phosphatidylcholine and phosphatidylserine (4:1 mol/mol) were examined by 2H nuclear magnetic resonance (NMR). Dipalmitoylphosphatidylcholine deuterated at the alpha- and beta-positions of the choline moiety was used to probe the surface region of the membranes. Addition of each DAG except DP caused a continuous decrease in the beta-deuteron quadrupole splittings and a concomitant increase in the alpha-deuteron splittings indicating that DAGs induce a conformational change in the phosphatidylcholine headgroup. Additional evidence of conformational change was found at high DAG concentrations (> or = 20 mol%) where the alpha-deuteron peaks became doublets indicating that the two alpha-deuterons were not equivalent. The changes induced by DP were consistent with the lateral phase separation of the bilayers into gel-like and fluid-like domains with the phosphatidylcholine headgroups in the latter phase being virtually unaffected by DP. The DAG-induced changes in alpha-deuteron splittings were found to correlate with DAG-enhanced protein kinase C (PK-C) activity, suggesting that the DAG-induced conformational changes of the phosphatidylcholine headgroups are either directly or indirectly related to a mechanism of PK-C activation. 2H NMR relaxation measurements showed significant increase of the spin-lattice relaxation times for the region of the phosphatidylcholine headgroups, induced by all DAGs except DP. However, this effect of DAGs did not correlate with the DAG-induced activation of PK-C.
1,2-Dipalmitoylphosphatidylcholine↗
Application of a membrane fusion assay for rapid drug screening.
PURPOSE: The purpose of this study is to develop an in vitro assay for screening drug and their effects on membrane fusion and lysis of intracellular organelles. METHODS: A 96-well microtiter-dish turbidimetric assay using membrane components of the eggs of sea urchins, a marine invertebrate, was applied to monitor granule fusion and/or lysis. RESULTS: Of 18 drugs screened, 16 had no effect. One antineoplastic drug, tamoxifen, disrupted intracellular membranes in a calcium independent manner. Taxol, another antineoplastic drug, specifically inhibited calcium triggered exocytosis. CONCLUSIONS: This assay is inexpensive, simple, rapid, and does not require the sacrifice of animal life. It has the potential to identify drugs that are membrane active, as well as those which specifically perturb events involved in the secretion process.
Animals↗
Lateralization of membrane-associated protein kinase C in rat piriform cortex: specific to operant training cues in the olfactory modality.
Rats were trained on an olfactory and a control modality (auditory or visual) discrimination task and brain membrane-associated protein kinase C (mPKC) was subsequently assessed using quantitative autoradiography of radiolabelled phorbol ester binding. In rats which received olfactory-cued training, mPKC showed a highly significant lateralization in the piriform cortex but not in the hippocampus. Both olfactory-trained rats and control modality rats showed a significant increase in mPKC in the hippocampus when compared to naive rats. Thus, while behavioral training procedures appeared to result in a hippocampal increase in the activated state of this enzyme as has been reported elsewhere, only olfactory learning produced an piriform cortex lateralization in the activated state of the enzyme. While the functional significance of such a change in the distribution of protein kinase C is still unclear, it does suggest that the monitoring of this enzyme's activational state may prove to be a useful tool in the study of memory formation in a wide variety of behavioral contexts.
Animals↗
Incorporation of fluorescent lipids into living rabbit hippocampal and cerebellar slices.
Incorporation of exogenously applied fluorescent lipids into living cells was exploited to probe cellular structure and function in living hippocampal and cerebellar slices as assessed by fluorescent imaging techniques and intracellular recording. Nitrobenzoxadiole-phosphatidylcholine (NBD-PC) and BODIPY phorbol ester, in vitro substrates of phospholipase activity and protein kinase C, respectively, were incorporated and distributed into specific cell populations. In the hippocampal slice, both probes labeled the somata and proximal dendrites of pyramidal and granule cells but were hetrogeneously distributed across the different hippocampal fields. Changes in fluorescent properties of NBD-PC in individual pyramidal cell and granule cell somata were quantified upon challenge with a muscarinic agonist known to modulate phospholipase A2 activity. In the cerebellar slice, both probes labeled Purkinje cell bodies and dendrites but only NBD-PC labeled stellate and granule cells. The cellular and functional specificity of these fluorescent lipid probes shows great promise for monitoring biochemical events in complex neuronal systems with significant spatial and temporal resolution.
4-Chloro-7-nitrobenzofurazan↗
Arachidonic acid and diacylglycerol ACT synergistically through protein kinase C to persistently enhance synaptic transmission in the hippocampus.
In model membranes, arachidonic acid and diacylglycerol have been proposed to synergistically induce a membrane-inserted, constitutively active form of protein kinase C. We have investigated the effects of these lipid protein kinase C activators on synaptic efficacy in the Schaffer collateral input to CA1 hippocampal pyramidal cells. Arachidonic acid (5 microM) perfusion combined with repetitive afferent stimulation had no consistent effect on field excitatory postsynaptic potentials recorded in stratum radiatum, while treatment with a cell-permeable diglyceride, oleoyl-acetylglycerol (5 micrograms/ml), followed by stimulation, led to a short-term potentiation. By contrast, the combination of oleoyl-acetylglycerol and arachidonic acid gave rise to a long-lasting non-decremental potentiation of field excitatory postsynaptic potentials. The induction of potentiation was "activity dependent", as there was either no significant effect or there was a measurable depression when repetitive synaptic stimulation was omitted. Furthermore, consistent with a protein kinase C-dependent process, the potentiation was blocked by the kinase inhibitors H-7 and staurosporine. The results suggest that relatively low concentrations of arachidonic acid and diacylglycerol work synergistically through protein kinase C to persistently enhance synaptic transmission. This synergy has the makings of an associative (Hebbian) device for long-term potentiation induction operating at the second messenger level.
Animals↗
Effects of diacylglycerols and Ca2+ on structure of phosphatidylcholine/phosphatidylserine bilayers.
The combined effects of the diacylglycerols (DAGs) with the various acyl chains and Ca2+ on the structure of phosphatidylcholine/phosphatidylserine (4:1 mole/mole) bilayers were studied using 2H- and 31P NMR. The following DAG- and Ca(2+)-induced bilayer perturbations were identified. 1) Increased tendency to form nonbilayer lipid phases was induced by diolein or stearoylarachidonoylglycerol, and was synergistically enhanced by the addition of Ca2+. 2) "Transverse" bilayer perturbation was induced by dioctanoylglycerol. The addition of this DAG caused increased ordering of the phospholipid acyl side chains in the region adjacent to the headgroup, with the concomitant decrease of the order toward the bilayer interior. 3) Separation of the phosphatidylcholine and phosphatidylserine bilayer components was induced by combinations of relatively high (1:5 mole/mole to phosphatidylserine) Ca2+ and 25 mol% (to the phospholipids) of diolein, stearoylarachidonoylglycerol, or oleoylacetylglycerol. 4) Lateral phase separation of the bilayers on the regions of different fluidities was induced by dipalmitin. These physicochemical effects were correlated with the effects of these DAGs and Ca2+ on the activity of protein kinase C. The increased tendency to form nonbilayer lipid phases and the transverse bilayer perturbations correlated with the increased protein kinase C activity, whereas the actual presence of the nonbilayer lipid phases, as well as the separation of the phosphatidylcholine and phosphatidylserine components, was associated with the decrease in the protein kinase C activity. The lateral phase separation of the bilayer on gel-like and liquid crystalline regions did not have an effect on the activity of the enzyme. These results demonstrate the importance of the physicochemical properties of the membranes in the process of activation of protein kinase C.
Biophysical Phenomena↗
Associative learning potentiates protein kinase C activation in synaptosomes of the rabbit hippocampus.
Using electrophysiological, biochemical, and autoradiographic techniques, changes in protein kinase C (PKC) activity in specific regions of the hippocampus have been previously implicated in classical conditioning of the nictitating membrane response of the rabbit. Here we report that activation of PKC is potentiated 2- to 3-fold in synaptosomes of the hippocampal CA1 and CA2 to -3 regions in rabbits that have undergone classical conditioning of the nictitating membrane response. This potentiation is apparently due to a change in the biochemical properties of PKC within the synaptosomes and is not a result of an increase in total PKC activity. This observation correlates a subcellular biochemical change with classical conditioning of a mammal.
Animals↗