David S. Lester

Arachidonic acid and diacylglycerol act synergistically to activate protein kinase C in vitro and in vivo

Using a well-defined model membrane bilayer system, incorporation of both lipid second messengers, 1,2-diacylglycerol and arachidonic acid, at submaximal activating concentrations, resulted in a synergistic activation of protein kinase C in a Ca2+/phosphatidylserine-dependent manner as measured by monitoring phosphorylation of phosphoprotein substrates. The arachidonic acid appears to modulate membrane properties both at the hydrocarbon core and the membrane surface increasing the availability of the diacylglycerol which can bind to and subsequently activate the enzyme. Co-application of these two lipid activators to the Hermissenda photoreceptor reduced K+ channel conductance in a synergistic manner via a PKC-dependent pathway. Thus, these in vivo and in vitro studies suggest that the membrane bilayer properties of these PKC lipid activators interact to specifically regulate the cellular lipid microenvironment resulting in PKC activation.

Cell Specificity of Molecular Changes During Memory Storage

Abstract: The aeolid nudibranch, Hermissenda crassicornis, exhibits Pavlovian conditioning to paired light and rotational stimuli and it has been suggested that protein kinase C (PKC) may play a critical role in the cellular mechanism for this conditioned behavioral response in the B‐cell photoreceptor. The present study was designed to further examine learning‐specific PKC involvement in identified cellular areas, particularly those in the visual‐vestibular network, of the Hermissenda nervous system after Pavlovian conditioning. As used in previous vertebrate studies, the highly specific PKC radioligand, [3H]phorbol‐12,13‐dibutyrate ([3H]‐PDBU), was used to determine the binding characteristics of the molluscan protein receptor considered to be PKC. The binding was specific, saturable, and could be displaced by a soluble diacylglycerol analogue. The binding activity was distributed evenly between the cytosol and the membrane. All of these analyses suggest that [3H]PDBU binds primarily to PKC in Hermissenda as it does in many other systems. Computerized grain image analysis was then used to determine the cellular localization of PKC as a function of Pavlovian conditioning. The medial and intermediate B photoreceptor and the optic ganglion showed significantly increased [3H]PDBU binding in conditioned animals. The present results provide the first report of an associative learning change of a key signal transduction component in identified neurons.

Infrared Spectroscopic Imaging of the Biochemical Modifications Induced in the Cerebellum of the Niemann-Pick type C Mouse.

We have applied Fourier transform infrared (IR) spectroscopic imaging to the investigation of the neuropathologic effects of a genetic lipid storage disease, Niemann-Pick type C (NPC). Tissue sections both from the cerebella of a strain of BALB/c mice that demonstrated morphology and pathology of the human disease and from control animals were used. These samples were analyzed by standard histopathological procedures as well as this new IR imaging approach. The IR absorbance images exhibit contrast based on biochemical variations and allow for the identification of the cellular layers within the tissue samples. Furthermore, these images provide a qualitative description of the localized biochemical differences existing between the diseased and control tissue in the absence of histological staining. Statistical analyses of the IR spectra extracted from individual cell layers of the imaging data sets provide concise quantitative descriptions of these biochemical changes. The results indicate that lipid is depleted specifically in the white matter of the NPC mouse in comparison to the control samples. Minor differences were noted for the granular layers, but no significant differences were observed in the molecular layers of the cerebellar tissue. These changes are consistent with significant demyelination within the cerebellum of the NPC mouse.

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 enzymes activational state may prove to be a useful tool in the study of memory formation in a wide variety of behavioral contexts.

Chapter 15: Activation of protein kinase C phosphorylation pathways: a role for storage of associative memory

Publisher Summary To study neuronal changes that store associative memory, Pavlovian conditioning was demonstrated in a snail preparation where it was possible to precisely trace the flow of information through well-defined neuronal networks that mediate the learning. This animal can be taught to associate a light stimulus with vestibular stimulation. The excitable state in identified photoreceptors of the eye is elevated following conditioning due to persistent changes in the molecular regulation of specific potassium currents for 14 days or longer. In a second system, rabbits are conditioned to associate an auditory tone with a puff of air to the surface of the eye causing the nictitating membrane of the eye to extend on subsequent days in response to the tone alone. Memory-specific changes in ion-channel properties and their molecular basis, similar to those observed in Hermissenda , have been measured in the CA1 pyramidal cells of the rabbit hippocampus. The parallels between mechanisms of memory storage in these and other model preparations suggest conserved molecular mechanisms of memory storage.

Application of a Membrane Fusion Assay for Rapid Drug Screening

AbstractPurpose. 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.

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.

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.

Heterogenous Distribution of Fluorescent Phorbol Ester Signal in Living Sea Urchin Embryos

The introduction of isolated genes or recombinant DNA into cultured cells is a key procedure in cell and molecular biological research. At present there are many methods for gene transfer. All of these methods have certain shortcomings. Most of them are strongly cell-type dependent (i.e., they can work only on certain cells), and the transfer efficiency is frequently unsatis- factorily low. Furthermore, these methods often produce un- desirable biological or chemical side-effects. Recently, a new physical method has been developed ( 1). The cell membrane can be temporarily permeabilized by exposing the cell to a pulse of high intensity electric field (2). This process is attributed to the creation of resealable pores in the cell mem- brane and is thus called “electroporation.” The electroporation method offers more advantages than most other methods. First, it is simple to use and is time efficient. It can also be used to inject a single cell or millions of cells. Second, because it is a physical method, electroporation is less dependent on cell type. Third, it has fewer harmful biological and chemical side effects. In the last several years, the electroporation method has been successfully used to introduce cloned genes into a wide variety of cells, including mammalian cell lines, isolated cells, plant cells, bacteria, and yeast (3, 4). In addition to gene transfer, electroporation has the potential for use as a micro-injection method to introduce molecules other than DNA, such as second messengers, kinases, and kinase in- hibitors, drugs, and antibodies into a large number of cells. We are currently working to improve this technology. Our approach is to experiment with different wave forms and pulse protocols and to test their effects on cell survival rate and transfection efficiency. Our earlier results had suggested that, by

Applications of Fourier transform infrared imaging microscopy in histopathology

Infrared microspectroscopic imaging has the capabilities of generating spatial information as well as the standard spectroscopy. By taking advantage of the vibrational spectral signatures of biological components, such as lipids and proteins, one has have the capability of obtaining images of the intrinsic distribution of these molecules. The authors have applied this approach to analyze tissue sections of biomedical relevance in order to determine the potential of this application as an alternate approach histopathology. The advantage of this technique is that tissue need not be fixed or stained, contrast obtained from the intrinsic distribution of the biomolecular components. This technique provides considerable application for many histopathological applications and could result in the significant increase in the speed and resolution of pathological analyses, such as biopsies.