#### Graeme

##### Vampyroteuthis
Registered
I was wondering if anyone knew of any membrane adaptations in deep-sea cephalopods? Examples would be temperature, pressure etc?
I think it's cell membrane adaptations as opposed to larger mebranes.

Graeme

#### Steve O'Shea

##### TONMO Supporter
Supporter
Nope (as in not aware), but I'd be surprised if anyone had actually looked at it! Sounds interesting!

#### Graeme

##### Vampyroteuthis
Registered
Hmmm, it's funny how Ceph's seem to generate a lot of interest, but when it actually comes down to studying them, there's not a lot of stuff compared to other animals. Mind you I s'pose it leaves a lot of niches open... Imagine doing that for a PHD though!! That would be pretty cool.

Graeme

#### Jean

##### Colossal Squid
Supporter
I think one of the reasons they are not studied as much as other animals, is that they are difficult to get hold of (and expensive,boats, special capture gear etc etc) and can be extremely difficult to keep alive especially the open ocean/deepwater types! But there are some intrepid souls out there who are trying.......like our own Dr SOS and TTF!

J

#### Graeme

##### Vampyroteuthis
Registered
... I'll do it! I'm stupid enough to be thrown on a boat in the middle of an Arctic Winter all in the name of fame and fortune... I mean Science!
Seriously, I think it would be quite interesting, but I'd need a microbiologist to translate everything everything for me- I get about as far as "now the cell membrane is..." before "zzzzzzzzzzzzzzz" or "...... "

Graeme

#### Feelers

##### Vampyroteuthis
Registered
Grab a squid research paper and read through the references. Then read the references of those. There are a quite a lot of papers out there, problem is they arent exaclty "user friendly" in the casual reading sense.

I wouldnt have a clue if there's anything about cell membranes - but there was a hell of a lot of references to see, pages and pages, so you might get lucky.

I've done a little microbio - most papers generally have a breif rundown of how things work -its when they give all the data that things get messy.

#### Fujisawas Sake

##### Larger Pacific Striped Octopus
Supporter
This is a good question... is there a major membrane morphology difference between deep sea and shallower-water cephalopods? If so, why?

#### Steve O'Shea

##### TONMO Supporter
Supporter
Hmmmmmmmmmmmmm

Found the following, using "squid cell membrane" as keywords:
........................
Selective open-channel block of KV1 potassium channels by S-nitrosodithiothreitol (SNDTT)
by Brock, Mathew William, Ph.D., Stanford University, 2003, 214 pages; AAT 3085165
School: Stanford University
School Location: United States -- California
Index terms(keywords): Open-channel block, Potassium channels, Nitrosodithiothreitol-S, Voltage-gated potassium channels
Source: DAI-B 64/03, p. 1109, Sep 2003
Source type: DISSERTATION
Subjects: Neurology, Biophysics, Organic chemistry
Publication Number: AAT 3085165
Document URL: http://proquest.umi.com.ezproxy.aut...1&sid=1&Fmt=2&clientId=7961&RQT=309&VName=PQD
ProQuest document ID: 765393901

Abstract (Document Summary)

Blockade of voltage-gated K + (Kv) channels is a feature of many large quaternary and tertiary amines. These compounds bind with a 1:1 stoichiometry in an aqueous cavity along the channel pore that is exposed to the cytoplasm only when channels are open. This thesis addresses the action of S-nitrosodithiothreitol (SNDTT; ONSCH 2 CH(OH)CH(OH)CH 2 SNO), which produces qualitatively similar "open-channel block" in Kv channels despite its unconventional (small, electrically neutral, and polar) structure. In whole-cell voltage-clamped squid giant fiber lobe neurons, bath-applied SNDTT causes reversible time-dependent block of delayed-rectifier K + channels, but not Na + or Ca 2+ channels. The inactivation-removed Shaker B (ShBΔ) Kv1 channel expressed in HEK 293 cells is blocked in a similar manner and was used to further characterize the action of SNDTT. Dose-response data for ShBΔ indicate that two molecules of SNDTT can bind to each open channel, but binding of a single molecule is sufficient for block. The dissociation constant for the second molecule bound (0.14 mM) is lower than for the first (0.67 mM), indicating cooperativity. Surprisingly, the steady-state level of block by this electrically neutral compound has a voltage-dependence (∼-0.25 e 0 ) similar in magnitude but opposite in directionality to that reported for amines. Both nitrosyl (-NO) groups on SNDTT (one on each sulfur atom) are required for block, but transfer of these reactive groups to channel cysteine residues is not involved. Competition with internal tetraethylammonium indicates that bath-applied SNDTT crosses the cell membrane to act at an internal site. Through targeted mutagenesis, we have identified two contiguous residues (Thr469 and Ile470) in the channel cavity that are strong determinants of SNDTT sensitivity. At position 469, a side chain -OH group is required for high affinity block, and may form a hydrogen bond with an -NO group on SNDTT. Finally, SNDTT is remarkably selective for Kv1 subfamily channels. When individually expressed in HEK 293 cells, rat Kv1.1-1.6 display profound time-dependent block by SNDTT, an effect not seen for rat Kv2.1, 3.1, or 4.2. SNDTT may therefore be useful as a pharmacological probe of Kv subtype, and may represent the prototype for a new class of pharmaceuticals selectively targeting Kv1 channels.
...................................
Nanoparticles: Engineering, assembly, and biomedical applications
by Kim, Do-Kyung, Ph.D., Kungliga Tekniska Hogskolan (Sweden), 2002, 216 pages; AAT C809956
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School: Kungliga Tekniska Hogskolan (Sweden)
School Location: Sweden
Index terms(keywords): Nanoparticles, Biomedical, Ferrofluids, Superparamagnetism
Source: DAI-C 63/04, p. 854, Winter 2002
Source type: DISSERTATION
Subjects: Materials science
Publication Number: AAT C809956
ISBN: 9172832657
Document URL: http://proquest.umi.com.ezproxy.aut...1&sid=1&Fmt=2&clientId=7961&RQT=309&VName=PQD
ProQuest document ID: 725953091

Abstract (Document Summary)

This thesis deals with novel aspects of Nanotechnology through " bottom-up " and " top-down " strategies, applied to biotechnology as an interdisciplinary study.

The main objectives of this thesis are to design SPION (superparamagnetic iron oxide nanoparticles) surface modified with other biocompatible agents, varying from organic to polymer and biocompatible materials, such as proteins. The particles have been introduced to intact organs of living animals (rat brain) to examine how they interact in the brain tissue and to confirm the feasibility of using SPION for biomedical applications such as MR imaging.

Several different types of materials including SPION (first generation), immobilized biocompatible materials on SPION (second generation), for in-vivo biomedical applications, nanowires, nanotubes have been prepared by using different aspects of Nanotechnology. Various processes and techniques for the preparation of functional nanomaterials such as coprecipitaion, microemulsion (μE), and template-assisted electrodeposition are developed.

Core-shell structure nanocomposites are fabricated by template-directed self-assembly ( bottom-up ). Controlled electroless deposition of Au is followed by a subsequent removal of the template core without destroying the formed Au shells. The work also includes the development of microcontact printing (μCP) techniques, where the ink used on the surface of the stamp is made of aminopropyl trimethoxy silane (APTMS). The approach is demonstrated with the formation of 2D and 3D structures.

Several different types of magnetic measurements of SPION are investigated. Authentication of superparamagnetism has been carried out by SQUID measurements, up to 7 Tesla, and evaluating the basic physical properties by the Langevien theory. Electron spin resonance (ESR) measurements have been performed as a function of temperature with different particle sizes. The line width of the ESR spectra can be correlated to the distribution of the SPION exchange interactions. Microwave energy absorption rates of SPION have been calculated using a non-linear fitting to experimental data.

The in-vivo experiments showed that, after injection of starch coated SPION into rat brain parenchyma in striatum, a strong phagocytic uptake of the particles is observed due to their strong affinity to the body cell. Diffusion barriers between blood and neural tissue, in the endothelium of the parenchymal vessels (BBB), in the epithelia of the chroid plexuses, and arachnoid membrane (blood-CSF barriers), severely restrict penetration of several diagnostic agents.

The prediction of SPION transport has been made by the modeling of the movement of a single SPION in a biological capillary system. The model considered the four most important factors, i.e. particle size, capillary diameter, distance between the magnets, and capillary length.
.....................................

#### Steve O'Shea

##### TONMO Supporter
Supporter
Mechanisms of synaptic vesicle endocytosis: The functions of clathrin in the nerve terminal
by Morgan, Jennifer R., Ph.D., Duke University, 2001, 263 pages; AAT 3057435
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School: Duke University
School Location: United States -- North Carolina
Index terms(keywords): Synaptic vesicle, Endocytosis, Clathrin, Nerve terminal
Source: DAI-B 63/06, p. 2738, Dec 2002
Source type: DISSERTATION
Subjects: Neurology, Cellular biology
Publication Number: AAT 3057435
ISBN: 0493727124
Document URL: http://proquest.umi.com.ezproxy.aut...1&sid=1&Fmt=2&clientId=7961&RQT=309&VName=PQD
ProQuest document ID: 727313791

Abstract (Document Summary)

Clathrin-coated vesicles (CCVs) mediate membrane budding and recycling in all cell types. CCVs are formed when clathrin and other accessory proteins are recruited to the donor membrane and assemble together into a proteinaceous coat, thereby inducing the invagination and pinching off of a small vesicle coated with these proteins. Following CCV formation, clathrin must be uncoated from the vesicle before the vesicle can be re-used. Although some of the proteins involved in CCV formation and uncoating have been identified, the precise molecular mechanisms underlying clathrin-mediated endocytosis are not well understood.

In neurons, CCVs are involved in synaptic vesicle recycling under conditions of heavy stimulation. However, whether CCVs participate in synaptic vesicle recycling under more physiological levels of synaptic activity is not known. Therefore, I examined the role of CCVs in synaptic vesicle recycling under low levels of synaptic activity, and I examined molecular mechanisms of CCV formation and uncoating in nerve terminals. To do so, I first identified peptides or protein fragments that inhibited clathrin assembly or uncoating in vitro . Then, I injected these inhibitory reagents into squid giant presynaptic terminals and tested for their effects on synaptic transmission and CCV formation and uncoating in vivo at 0.03 Hz stimulation.

My results indicate that the clathrin assembly activity of the synapse-specific clathrin assembly protein (AP), AP180, is essential for synaptic vesicle endocytosis. In addition, my results show that the structurally-diverse monomeric AP180 and the tetrameric AP-2 assemble clathrin by virtue of possessing multiple copies of a DLL tripeptide motif. These results allowed me to propose a novel model for CCV formation in which APs assemble clathrin by cross-linking several clathrin molecules together. Finally, my results provide the first evidence that Hsc70 and auxilin, as well as their interaction at auxilin's well-conserved HPD tripeptide motif, are essential for CCV uncoating in vivo and synaptic vesicle endocytosis. Together, my results indicate that clathrin functions are essential for synaptic vesicle endocytosis under physiological levels of nerve activity. In addition, my results provide novel details to the mechanisms of CCV formation and uncoating that can be extrapolated to clathrin-mediated endocytosis in all cell types.
....................................
The cellular and biochemical environment of the symbiotic light organ of the Hawaiian squid Euprymna scolopes
by Nyholm, Spencer Victor, Ph.D., University of Hawai'i, 2001, 130 pages; AAT 3030190
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School: University of Hawai'i
School Location: United States -- Hawaii
Index terms(keywords): Symbiotic, Light organ, Euprymna scolopes, Vibrio fischeri
Source: DAI-B 62/10, p. 4415, Apr 2002
Source type: DISSERTATION
Subjects: Zoology, Microbiology
Publication Number: AAT 3030190
ISBN: 0493427996
Document URL: http://proquest.umi.com.ezproxy.aut...1&sid=1&Fmt=2&clientId=7961&RQT=309&VName=PQD
ProQuest document ID: 726046171

Abstract (Document Summary)

The light organ symbiosis between the Hawaiian bobtail squid Euprymna scolopes and the bioluminescent bacterium Vibrio fischeri provides the opportunity to study the influence of beneficial bacteria on animal tissues. The extracellular bacterial symbionts are housed in epithelia-lined crypt spaces in the host's light organ. The host provides the symbionts with nutrients while the bacteria provide light for the host that is likely used in the squid's nocturnal foraging behavior. The association is very specific and begins within hours after hatching, when V. fischeri from the environment colonize the squid host. The goal of this dissertation was to characterize the microenvironment surrounding the symbiont population from the earliest onset of colonization of the juvenile squid to the adult animal. Within 1 h after hatching, ciliated host cells on the surface of the light organ secreted mucus and created currents that entrained Gram-negative bacteria in dense aggregates outside the light organ and near pores that lead to sites of colonization. The bacterial membrane component peptidoglycan was shown to induce mucus secretion. Bacterial aggregation, and subsequent migration and colonization of the light organ, proceeded with an increased specificity for the symbiont V. fischeri . Experimental manipulation of the host provided evidence that once colonized, the host undergoes a diel behavior in which 95% of the symbionts are vented each morning. The ventate emerged as a thick exudate that contained a mixture of V. fischeri and host cells, a subpopulation of which were squid hemocytes, all embedded in a thick protein-rich matrix. The soluble proteins in the light organ environment changed on a diel rhythm, as did the host epithelium, which exhibited cell shedding during the day. Finally, hemocytes of adult hosts were capable of binding to a variety of bacteria with different affinities. Adhesion of these cells to V. fischeri changed when the light organs were cured with antibiotics, suggesting that the symbiosis alters the behavior of host hemocytes. Together these studies add to the knowledge of how symbiotic environments are created and maintained.

#### Steve O'Shea

##### TONMO Supporter
Supporter
Characterization of squid KV1 voltage-gated potassium channels by expression in Xenopus oocytes
by Liu, Taylor I-Tso, Ph.D., Stanford University, 2000, 191 pages; AAT 9961919
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School: Stanford University
School Location: United States -- California
Index terms(keywords): Squid, KV1, Voltage-gated, Potassium channels, Oocytes, Xenopus
Source: DAI-B 61/02, p. 716, Aug 2000
Source type: DISSERTATION
Subjects: Neurology, Cellular biology
Publication Number: AAT 9961919
ISBN: 0599659203
Document URL: http://proquest.umi.com.ezproxy.aut...1&sid=1&Fmt=2&clientId=7961&RQT=309&VName=PQD
ProQuest document ID: 731864011

Abstract (Document Summary)

Studies of voltage-gated sodium and potassium channels in squid giant axons have founded our understanding of action potential propagation. Cloning α-subunits of voltage-gated potassium channels (SqKv1) and the putative sodium channel (GFLN1) from the squid stellate ganglion (SG) has allowed us to study these ion channels at the molecular and cellular level. We first determined cell-type specificity of expression for GFLN1 and four SqKv1 cDNA clones (SqKv1 A-D) within the squid SG. SqKv1 A-D differ in N-terminal length and in isolated amino acid residues in the tetramerization domain (T1) and C-terminus. We characterized differences in properties among SqKv1 channels by expression in Xenopus oocytes, and explored the relationship between primary structure and functional variation.

In situ hybridization was used to localize GFLN1 mRNA to neurons with large or long axons. GFLN1-specific cRNA probes labeled both giant fiber lobe (GFL) neurons and large neurons in the cellular layer of the main stellate ganglion. In contrast, SqKv1A is prominently expressed in the GFL and not in the main SG. SqKv1B exhibits the opposite pattern, localizing to the main SG. SqKv1D was only expressed in SG at low levels, and SqKv1C expression was not detectable.

Heterologous expression of SqKv1 A-D in Xenopus oocytes and biophysical analysis by cell-attached patch clamp and two-microelectrode voltage clamp revealed differences in functional expression levels among SqKv1 variants. SqKv1D expressed ∼10-fold more current than SqKv1B and ∼80-fold more current than SqKv1A. Using site-directed mutagenesis we determined that low expression level of SqKv1A is attributable to the distal N-terminus (first 11 residues) and one residue in the T1 domain. In contrast, a single amino acid difference in the T1 domain accounts for the expression level difference between SqKv1B and SqKv1D.

Differences in functional expression level were also accompanied by differential glycosylation states among SqKv1 variants. High functional expression levels appeared to correlate with presence of channels containing complex-processed oligosaccharides. However, glycosylation was determined to be unnecessary for expression of functional channels on the cell surface. Nonetheless, analysis of glycosylation status may indicate the maturation state of SqKv1 channels within the membrane protein processing pathway and provide a biochemical correlate to the functional analyses.
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Induction of ouabain-sensitive channel activity by acid pH
by Khater, Kevin, Ph.D., The Herman M. Finch University of Health Sciences - The Chicago Medical School, 1999, 90 pages; AAT 9945848
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School: The Herman M. Finch University of Health Sciences - The Chicago Medical School
School Location: United States -- Illinois
Index terms(keywords): Transient currents, Sodium,potassium-ATPase, Ouabain-sensitive, Channel activity, Acid pH
Source: DAI-B 60/09, p. 4465, Mar 2000
Source type: DISSERTATION
Subjects: Biophysics
Publication Number: AAT 9945848
ISBN: 0599476257
Document URL: http://proquest.umi.com.ezproxy.aut...1&sid=1&Fmt=2&clientId=7961&RQT=309&VName=PQD
ProQuest document ID: 730183181

Abstract (Document Summary)

The effect of varying external pH on pre-steady state charge movement and steady state current mediated by the Na + /K + -ATPase was investigated in South African clawed toad ( Xenopus laevis ) oocytes. The experiments focused on determining the characteristics of a ouabain-sensitive inward current induced by acid pH and seen in external K + -free solutions [1] [2-4]. Five experimental techniques were used to study this inward current in Xenopus oocytes. (1) The two-microelectrode voltage clamp technique was used to determine the effect of changes in external [Na + ] on the ouabain-sensitive, current under K + -free external conditions at pH 5.6. High external [Na + ] increased the inward current at moderate membrane potentials (V m more positive than about -60 mV) and inhibited the current at membrane potentials more negative than about -80 mV. (2) The cut-open oocyte vaseline seal technique was used to determine the effects of external acidification on presteady-state transient currents under 3Na + /3Na + exchange conditions. At pH 5.6, an inward current is observed that increased in magnitude when pH was lowered to 4.6 and then 3.6. (3) Simultaneous current and flux measurements in squid ( Loligo peali ) giant axons were performed to determine the characteristics of H 2 DTG-sensitive 22 Na efflux and current at acidic external pH in external Na + - and K + -free solutions. The results showed that at pH 7.7, there is a Na + /K + -ATPase mediated (H 2 DTG-sensitive) 22 Na + efflux (3.8 pmol cm -2 s -1 ) present that increases in magnitude when external pH is lowered to 6.6 (6.8 pmol cm -2 s -1 ). (4) The cell-attached patch clamp technique with a pipette perfusion system was used to observe ouabain-sensitive single channel activity induced by acidic pH. The channel activity had a single channel conductance of 10.5 pS ± 0.0004, a mean open time of 8.05 msec ± 0.99, a mean short closed time constant of 1.93 msec ± 0.14, and a mean long closed time constant of 1220 msec ± 860 (at -100 mV and 100 mM external [Na + ]). A single binding site noncompetitive Na + inhibition model of permeation described the data. We, therefore, conclude that low pH induces a channel-like conformation of the Na + /K + pump.

#### Steve O'Shea

##### TONMO Supporter
Supporter
The assembly of intermediate filament networks
by Prahlad, Veena, Ph.D., Northwestern University, 1999, 231 pages; AAT 9953362
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School: Northwestern University
School Location: United States -- Illinois
Index terms(keywords): Filament networks, Cytoskeleton, Microtubules, Vimentin, Nestin
Source: DAI-B 60/12, p. 5876, Jun 2000
Source type: DISSERTATION
Subjects: Cellular biology, Biochemistry
Publication Number: AAT 9953362
ISBN: 0599565993
Document URL: http://proquest.umi.com.ezproxy.aut...1&sid=1&Fmt=2&clientId=7961&RQT=309&VName=PQD
ProQuest document ID: 730788901

Abstract (Document Summary)

The factors regulating the in vivo organization of Intermediate Filament (IF) networks are largely unknown. Previous studies have shown that IF organization appears to be regulated, in part, by factors that affect the dynamics of IF, and the assembly and disassembly of its constituent polymers. In addition, there is evidence that the organization of IF also depends on the integrity of the two other major cytoskeletal systems, the microtubule (MT) and microfilament (MF) networks. The studies described in this thesis suggest a mechanism by which IF organization might be coupled to IF dynamics, through their interactions with MIT. This mechanism, studied mainly during IF assembly in BHK-21 fibroblasts, involves the MT dependent transport of precursors of vimentin IF networks, vimentin particles, to regions within cells where they appear to assemble into short vimentin filaments. A similar mechanism of MT-dependent transport of NF particles appears to occur in a cell type as varied as the giant axon of the squid, Loligo paelei . This suggests that the MT-dependent transport of IF particles is a general mechanism involved in the assembly and maintenance of all IF networks. The transport of the vimentin and NF particles occurs at rates of 0.5-1.0 μm/sec and appears to be mediated by a member of the conventional kinesin family of motor proteins. Thus, the MT- and kinesin-dependent transport of IF particles to regions requiring local assembly of IF provides a mechanism by which IF dynamics is coupled to IF assembly and organization. Since the vimentin particles are not membrane bound, these studies also provide evidence for the involvement of kinesin in the intracellular transport of cytoskeletal proteins.

A component of the vimentin particles and vimentin IF is the high molecular weight IF protein nestin. Our studies aimed at characterizing nestin suggest how the unique structural properties of nestin might allow it to regulate vimentin IF organization. Nestin appears to copolymerize with vimentin at low concentrations, and apparently inhibits vimentin polymerization at high concentrations. Thus, nestin appears to be capable of regulating the state of assembly of vimentin IF. In addition, our studies suggest that nestin interacts with MT. Both these functions of nestin appear to involve its unusually long C-terminal tail. The observations presented here suggest a mechanism by which nestin might be involved in regulating the conversion of vimentin particles to vimentin filaments and how this process might be related to interactions between IF and MT.
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Vesicular repair of axonal injury
by Eddleman, Christopher Sean, Ph.D., The University of Texas Graduate School of Biomedical Sciences at Galveston, 1999, 110 pages; AAT 9924628
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School: The University of Texas Graduate School of Biomedical Sciences at Galveston
School Location: United States -- Texas
Index terms(keywords): Calcium, Membrane repair, Vesicular repair, Axonal injury
Source: DAI-B 60/04, p. 1454, Oct 1999
Source type: DISSERTATION
Subjects: Neurology, Cellular biology, Biophysics
Publication Number: AAT 9924628
ISBN: 0599241985
Document URL: http://proquest.umi.com.ezproxy.aut...1&sid=1&Fmt=2&clientId=7961&RQT=309&VName=PQD
ProQuest document ID: 733976941

Abstract (Document Summary)

Elevated intracellular Ca 2+ concentration ([Ca 2+ ]i) and injury-induced vesicles have been implicated in the repair of axons, but their respective roles are currently unknown. By use of electrophysiological techniques and confocal microscopy, the roles of calcium and injury-induced vesicles in axonal repair were investigated. Giant axons from crayfish ( Procambaris clarkii ) and squid ( Loligo pealei ) were used in vitro to show that crayfish medial giant axons (CMGAs) did, but squid giant axons (SGAs) did not, functionally recover (repair) in physiological saline after injury. After injury of CMGAs, vesicles formed, migrated to the cut end, and co-localized with a physical barrier. These results strongly suggested that injury-induced vesicles mediated the repair of transected CMGAs.

Injury-induced vesicles were induced by a rise in [Ca 2+ ] i ([Special characters omitted.] 100μM). When Ca 2+ was dialyzed into intact axons, vesicles formed whereas elevation of [K + ] i was ineffective and both [Na + ] i and [Cl - ] i ([Special characters omitted.] 400mM) only produced very small vesicles. Therefore, vesiculation was very calcium dependent and did not proceed unless [Ca 2+ ] i was increased above 100μM.

Injury-induced vesicles formed via Ca 2+ -dependent endocytosis of the axolemma. These vesicles often associated or fused with other structures during their journey to the injury site where they accumulated and mediated a physical barrier.

In addition to the [Ca 2+ ] i dependence of injury-induced vesiculation, Ca 2+ played a role in axonal repair by activating calpain, a Ca 2+ -dependent cysteine protease. Inhibition of calpain prevented the repair of transected CMGAs whereas exogenous calpain facilitated the repair of SGAs, which did not repair in physiological saline. Inhibition of calpain did not prevent vesiculation or the migration of vesicles to the injury site but rather was effective in the aggregation of injury-induced vesicles at the injury site. Therefore, Ca 2+ -activated calpain was an essential requirement in the repair of giant axons.

Finally, the formation of a physical barrier after injury was gradual and the permeability of the barrier, as assessed by the entry of dye molecules of decreasing size, decreased with time during barrier formation. Topological models demonstrated how vesicles could mediate the formation of a barrier after injury. Vesicular mechanisms of repair after injury also apply to other cell types where vesicles form after injury and aggregate at the site of injury. Vesicular repair of cellular injury may be a common mechanism of most cells, but the details of the repair process probably depend on the type of injury, type of cell, and origin of vesicular membrane.

#### Steve O'Shea

##### TONMO Supporter
Supporter
Analysis of electrophysiological models of spontaneous secondary spiking and triggered activity
by Enns-Ruttan, Jennifer Sylvia, Ph.D., The University of British Columbia (Canada), 1998, 216 pages; AAT NQ34522
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School: The University of British Columbia (Canada)
Index terms(keywords): Action potential, Membrane depolarization, Electrophysiological, Spontaneous secondary spiking, Triggered activity
Source: DAI-B 59/12, p. 6345, Jun 1999
Source type: DISSERTATION
Subjects: Mathematics, Neurology, Anatomy & physiology, Animals
Publication Number: AAT NQ34522
ISBN: 061234522X
Document URL: http://proquest.umi.com.ezproxy.aut...1&sid=1&Fmt=2&clientId=7961&RQT=309&VName=PQD
ProQuest document ID: 733035311

Abstract (Document Summary)

We have examined two mathematical models describing the electrophysiology of a neuron and a cardiac cell, respectively, which exhibit an unusual response to high frequency stimulation. For certain parameter sets, both models behave qualitatively like the classic Hodgkin-Huxley equations for a squid giant axon; several brief depolarizing current pulses give rise to a corresponding number of action potentials followed by a return to rest. However, if the parameters are adjusted to reflect certain experimental conditions, a few "spontaneous" action potentials sometimes follow the directly induced action potentials. The number of spontaneous action potentials depends on the number and frequency of action potentials in the original spike train. Our objective was to gain a qualitative understanding of the mechanisms involved in this phenomenon and the effects of certain experimental interventions in promoting or suppressing the occurrence of the spontaneous action potentials.

We first studied the Kepler and Marder (KM) model of spontaneous secondary spiking in a crab neuron. Then we examined the DiFrancesco-Noble (DN) equations for a mammalian cardiac Purkinje fiber which can exhibit a behaviour analogous to spontaneous secondary spiking, referred to in cardiac literature as triggered activity. Using a combination of bifurcation analysis and numerical computation, we showed that spontaneous action potentials are likely to occur in both models when a critical bifurcation parameter is just to the left of a saddle-node of periodics (SNP) bifurcation. In the KM model, the neurotransmitter, serotonin, promoted spontaneous secondary spiking by shifting the bifurcation parameter closer to the SNP. Similarly, in the DN equations, application of digitalis increased the bifurcation parameter (intracellular [Na + ]) while high extracellular [Ca 2+ ] shifted the SNP to a lower value, both effects promoting triggered activity.

Both models consist of an excitable subsystem and another subsystem that can build up slowly in response to action potentials. Spontaneous action potentials result from the bidirectional feedback between the two subsystems. By simplifying the KM model, we showed that a 3D model can exhibit spontaneous action potentials and that while the shape of the action potentials is unimportant, the relative time constants of the two subsystems are crucial.
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Characterization and functional analyses of the kinesin light chains and light chain isoforms
by Stenoien, David L., Ph.D., The University of Texas Southwestern Medical Center at Dallas, 1996; AAT 0577289
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School: The University of Texas Southwestern Medical Center at Dallas
School Location: United States -- Texas
Source: DAI-B 57/04, p. 2287, Oct 1996
Source type: DISSERTATION
Subjects: Cellular biology
Publication Number: AAT 0577289
Document URL: http://proquest.umi.com.ezproxy.aut...1&sid=1&Fmt=2&clientId=7961&RQT=309&VName=PQD
ProQuest document ID: 742599861

Abstract (Document Summary)

The kinesin holoenzyme is a heterotetramer consisting of two heavy and two light chains. The kinesin heavy chains possess the mechanochemical activities of kinesin. The kinesin light chains have been proposed to act in binding kinesin to organelles, but the evidence for this idea is indirect. To determine the role of the kinesin light chains in binding to organelles and to explore other potential functions of the light chains, antibodies against conserved domains of the kinesin light chains were generated and tested for their ability to inhibit kinesin function. One of these antibodies, KLC-All, was found to be a potent inhibitor of organelle traffic in squid axoplasm. This antibody was generated against a peptide found in the highly conserved tandem repeats. In vitro experiments with KLC-All suggested that this antibody inhibited organelle transport by causing kinesin to dissociate from organelles and implicated the tandem repeats in binding to organelles. The ability of kinesin to bind to organelles suggests that a kinesin receptor exists on these organelles. The interaction of kinesin with a putative receptor, kinectin, was further characterized. Kinectin lacked properties expected of a kinesin receptor and the specificity of its interaction with kinesin was found to be questionable. Other attempts to identify potential kinesin receptors have met with little success. This failure may be due to the lack of a ubiquitous kinesin receptor present on all organelles. Many different classes of organelles have kinesin associated with their surfaces but they may share few integral membrane proteins which could serve as a common kinesin receptor. Light chain isoforms exist which could interact with different receptors on different organelles and target kinesin to these organelles. To explore potential functions of these isoforms, antibodies that specifically recognize these isoforms were generated. These antibodies demonstrated that the light chain isoforms have unique subcellular, tissue, and developmental distributions suggesting that light chain isoforms are important for the function of kinesin under different circumstances. Evidence is presented that different isoforms are necessary for the transport of certain types of organelles and that certain cell types require these isoforms to regulate organelle transport.

#### Steve O'Shea

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Molecular mechanisms of neurotransmitter release: The functions of Rab3a, Rabphilin and SNAP-25
by Burns, Marie Elizabeth, Ph.D., Duke University, 1996, 201 pages; AAT 9707749
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School: Duke University
School Location: United States -- North Carolina
Index terms(keywords): exocytosis
Source: DAI-B 57/10, p. 6108, Apr 1997
Source type: DISSERTATION
Subjects: Neurology, Cellular biology
Publication Number: AAT 9707749
ISBN: 059114963X
Document URL: http://proquest.umi.com.ezproxy.aut...1&sid=1&Fmt=2&clientId=7961&RQT=309&VName=PQD
ProQuest document ID: 739505051

Abstract (Document Summary)

Neurotransmitter release is a highly regulated process that serves as the basis of neuronal intercommunication. Transmitter is stored within synaptic vesicles, which undergo a cycle of reactions at the active zones of presynaptic cells. Many proteins are thought to regulate the passage of synaptic vesicles through each stage of this cycle. The goal of my thesis has been to determine the function of three presynaptic proteins (Rab3a, Rabphilin, and SNAP-25) in neurotransmitter release. Combining electrophysiology, fluorescence imaging, and electron microscopy at the squid giant synapse, I have perturbed the function of each of these proteins in turn and observed the effect of these manipulations on neurotransmitter release and synaptic vesicle cycling.

Microinjection of Mss4, a Rab GDP-GTP exchange protein, into the presynaptic terminal enhanced neurotransmitter release. In contrast; perturbation of Rabphilin function potently inhibited neurotransmitter release. Examination of the morphology of presynaptic terminals injected with Rabphilin reagents revealed that inhibition of neurotransmitter release was correlated with perturbation of both exocytotic and endocytotic membrane compartments, suggesting a multifunctional role for Rabphilin in neurotransmitter release. My interpretation of these results is that Rabphilin regulates the attachment of vesicles to the plasma membrane, a process that was also inhibited by SNAP-25 perturbation. Together, these experiments suggest a model where Rab3 and Rabphilin regulate synaptic vesicle docking and the assembly of the SNARE complex, which then primes synaptic vesicles for fusion.
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Transport of magnesium and calcium across the membrane of excitable cells
by De Santiago Gonzalez, Jaime, Ph.D., The Herman M. Finch University of Health Sciences - The Chicago Medical School, 1996, 188 pages; AAT 9704843
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School: The Herman M. Finch University of Health Sciences - The Chicago Medical School
School Location: United States -- Illinois
Source: DAI-B 57/09, p. 5514, Mar 1997
Source type: DISSERTATION
Subjects: Neurology, Biochemistry, Anatomy & physiology, Animals
Publication Number: AAT 9704843
ISBN: 0591114828
Document URL: http://proquest.umi.com.ezproxy.aut...1&sid=1&Fmt=2&clientId=7961&RQT=309&VName=PQD
ProQuest document ID: 739456651

Abstract (Document Summary)

The free intracellular concentration of Mg$\sp{2+}$ and Ca$\sp{2+}$, in excitable cells, is several fold lower than expected if they were distributed passively. The membrane transport mechanisms responsible to extrude these ions are studied in dialyzed squid axons and perfused barnacle muscle cells.

In this work has been shown that there is an absolute requirement of intracellular K$\sp+$ and Cl$\sp-$ for the extracellular Mg$\sp{2+}$-dependent Na$\sp+$ efflux and that removal of extracellular Mg$\sp{2+}$ produce a simultaneous and equimolar reduction in Na$\sp+$, K$\sp+$ and Na$\sp+$, Cl$\sp-$ efflux. These result suggest that the putative Na$\rm\sp+/Mg\sp{2+}$ exchanger may in fact be a $\rm Na\sp+K\sp+Cl\sp-/Mg\sp{2+}$ exchanger.

Systematic studies on Mg$\sp{2+}$ transport in excitable cells is hindered by the limited seasonal availability of squid and by the scarcity of the only useful radioactive Mg$\sp{2+}$ isotope, $\sp{28}$Mg (half life = 21 hrs and currently produced only one day a year). To overcome these limitations it has been shown: (1) the presence of Na$\sb0$-dependent Mg$\sp{2+}$ and Mn$\sp{2+}$ efflux, and (2) the presence of Mg$\sb0$-dependent Na$\sp+$ efflux in barnacle muscle cells.

It has been shown that the $\rm Na\sp+/Ca\sp{2+}$ exchanger can operate in its "$\rm Ca\sp{2+}$ efflux mode" in the complete absence of extracellular and intracellular K$\sp+$, indicating that K$\sp+$ is not co-transported with Ca$\sp{2+}$ in exchange for Na$\sp+$ as has been shown in retinal cells.

In barnacle muscle cells we need to increase Ca$\rm\sb{i}$ to activate the $\rm Na\sp+/Ca\sp{2+}$ exchanger, but this manipulation produces opening of non selective cation channels, increasing the membrane conductance and making difficult to voltage clamp the cell. In this work has been shown that $\alpha$-chymotrypsin activates all the modes of the $\rm Na\sp+/Ca\sp{2+}$ exchanger, eliminates the need to raise Ca$\rm\sb{i}$, and leaves an exchanger still subject to further modulation.

It has been shown that the other Ca$\sp{2+}$ extrusion mechanism, the sarcolemmal Ca$\sp{2+}$ ATPase, mediates an exchange of Ca$\sp{2+}$ for H$\sp+$. It was found that external acidification stimulates whereas alkalinization inhibits Ca$\sp{2+}$ efflux, and that this process is ATP dependent and inhibited by Ca$\sp{2+}$ ATPase blockers (Cd$\sp{2+}$, vanadate, and eosin).

#### Steve O'Shea

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Characterization of a uniquemRNA population present in the squid giant axon
by Chun, Jong Tai, Ph.D., University of Pittsburgh, 1995, 135 pages; AAT 9820024
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School: University of Pittsburgh
School Location: United States -- Pennsylvania
Index terms(keywords): Lologo pealii, axon cytoskeleton
Source: DAI-B 58/12, p. 6413, Jun 1998
Source type: DISSERTATION
Subjects: Neurology, Molecular biology
Publication Number: AAT 9820024
ISBN: 0591716526
Document URL: http://proquest.umi.com.ezproxy.aut...1&sid=1&Fmt=2&clientId=7961&RQT=309&VName=PQD
ProQuest document ID: 736841491

Abstract (Document Summary)

The prevailing view in neurobiology contends that axons are devoid of RNA and protein synthetic capacity. However, recent studies indicated that both vertebrate and invertebrate axons contain some RNA in a growing number of experimental systems. In squid (Loligo pealii), the giant axon contains polyribosomes and a heterogeneous mRNA population composed of more than 50 different messages, raising the possibility that a select subset of proteins may be locally synthesized in the axonal territory. In this study, some of the individual mRNAs present in the squid giant axon were identified and quantitated to evaluate the biological significance of the axonal mRNA population. The results showed that the squid giant axon contains mRNAs encoding a diverse class of proteins including cytoskeletal proteins ($\beta$-actin, $\beta$-tubulin, and NF proteins), microtubule-based molecular motors (kinesin and MAP H1), a metabolic enzyme (enolase), and a putative integral membrane protein (pA6). The results of the competitive RT-PCR analyses indicated that kinesin is the most prevalent message in the axon, whereas $\beta$-tubulin mRNA is most abundant in the giant fiber lobe (GFL), where the parental cell bodies are located. The absolute levels of the six mRNAs varied over a 20- and 40-fold range within the axon and GFL, respectively. When the levels in the axon were normalized with the corresponding levels in the GFL, kinesin showed 10 times higher value than enolase. As a whole, the relative abundance profile of mRNAs in the axon was not directly related to their corresponding levels in the GFL. Taken together, these observations suggest that key constituents of the axonal cytomatrix, transport system, and energy metabolism are locally synthesized in the axon, and that specific mRNAs are selectively and differentially transported into the axonal domain.
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Identification ofmss4, a mammalian guanine nucleotide exchange factor (GEF) for a subset of Rab GTPases
by Burton, Janet Lynn, Ph.D., Yale University, 1995, 127 pages; AAT 9541401
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School: Yale University
School Location: United States -- Connecticut
Source: DAI-B 56/08, p. 4095, Feb 1996
Source type: DISSERTATION
Subjects: Cellular biology, Molecular biology
Publication Number: AAT 9541401
Document URL: http://proquest.umi.com.ezproxy.aut...1&sid=1&Fmt=2&clientId=7961&RQT=309&VName=PQD
ProQuest document ID: 741101431

Abstract (Document Summary)

The presynaptic nerve terminal is filled with synaptic vesicles which contain non-peptide neurotransmitters whose regulated release is responsible for the rapid point to point communication between neurons. Although the synaptic vesicle lifecycle is unique to neurons, it has become increasingly clear that molecular mechanisms of intracellular membrane transport are conserved in all levels in the secretory pathway and in evolution, from yeast to neurons.

In order to isolate proteins involved in synaptic vesicle traffic or other vesicular transport pathways in mammalian cells, a genetic screen was undertaken to identify mammalian suppressers of yeast secretory mutants. Using this approach, we were able to identify a novel mammalian cDNA, mss4, which at the restrictive temperature, could restore both growth and secretion to sec4-8 cells, a yeast strain which harbors a point mutation in the small GTPase, Sec4. Amino acid sequence comparisons revealed that Mss4 had homology with the yeast protein, Dss4, which when mutated can suppress the sec4-8 strain. Biochemical analysis revealed that both Mss4 and Dss4 were able to promote GDP-release from Rab members but not Ras. These findings strongly suggest that the molecular mechanisms governing vesicular transport are conserved between yeast cells and neurons.

Further investigation of the Mss4 protein using gel overlays, filter assays which measure guanine nucleotide binding, and co-precipitation studies, demonstrated that Mss4 is not a general Rab accessory protein, but is highly selective for a subset of the Rab GTPases which include Rab1, Rab3 Rab8 and Rab10. In addition to stimulating GDP-release, Mss4 was found to promote the association of GTP selectively onto these Rabs. Mss4 exists in a complex with Rab3a but not Rab5 in brain extract, and guanine nucleotides disrupt the Rab3a-Mss4 interaction. Finally, Mss4 was shown to enhance neurotransmitter release when injected into the squid giant nerve terminal. Together, these data define Mss4 as a GEF for a subset of Rabs which influences multiple vesicular transport steps including synaptic vesicle exocytosis.

#### Steve O'Shea

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Analysis of cellular oscillators using sinusoidal forcing
by Gray, Richard Alan, Ph.D., University of Virginia, 1993, 116 pages; AAT 9402655
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School: University of Virginia
School Location: United States -- Virginia
Source: DAI-B 54/08, p. 4269, Feb 1994
Source type: DISSERTATION
Subjects: Biomedical research, Neurology
Publication Number: AAT 9402655
Document URL: http://proquest.umi.com.ezproxy.aut...1&sid=1&Fmt=2&clientId=7961&RQT=309&VName=PQD
ProQuest document ID: 744939631

Abstract (Document Summary)

We have developed a non-linear method to study membrane potential oscillations through the use of sinusoidal forcing. The protocol involves injecting a constant holding current across cell membranes to keep their membrane potentials at some pre-stimulus value and injecting sinusoidal current systematically varying the frequency. The resulting membrane potential signals were decomposed into their harmonic components. The amplitude and phase angle values of the first five harmonics were calculated as a function of the driving frequency.

We have termed this procedure the "resonance method" and tested it on two mathematical models: the standard Hodgkin-Huxley (1952) space-clamped squid axon model and a two-cell reciprocal inhibition model. For each model the amplitude of the first harmonic of membrane potential response to sinusoidal forcing exhibited a single "resonant" peak at the normal oscillation frequency. The higher harmonic values were greatest near the normal oscillating frequency for both models indicating that non-linearities are important in generating oscillations.

The resonance protocol was also applied to neurons involved in generating swimming activity in the medicinal leech. The procedure was applied to "oscillator" cells which are thought to be part of the "core oscillator" that generates swimming. The first harmonic amplitude response for these oscillator cells showed a resonant peak within the normal swimming frequency range (0.5 to 2.0 Hz). In addition the resonance method was applied to a motor neuron in the absence and presence of serotonin. The first harmonic amplitude response showed a resonant peak within the swimming range in the presence but not in the absence of serotonin. These results from both oscillator cells and a motor neuron indicate that the cells important in generating swimming activity show a resonant peak in their first harmonic response that occurs within the normal swimming frequency range.

We show that this resonance method provides a means for studying membrane properties and their relationship to oscillations in both individual cells and in cells imbedded in circuits.
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Association of kinesin with membrane-bounded organelles
by Leopold, Philip Lutz, Ph.D., The University of Texas Southwestern Medical Center at Dallas, 1993; AAT 0573193
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School: The University of Texas Southwestern Medical Center at Dallas
School Location: United States -- Texas
Source: DAI-B 54/04, p. 1822, Oct 1993
Source type: DISSERTATION
Subjects: Neurology
Publication Number: AAT 0573193
Document URL: http://proquest.umi.com.ezproxy.aut...1&sid=1&Fmt=2&clientId=7961&RQT=309&VName=PQD
ProQuest document ID: 747476421

Abstract (Document Summary)

The morphology of a typical neuron includes a cell body and one or more processes which have lengths many times the diameter of the soma, thus creating a unique set of problems relating to maintenance of important cellular specializations (such as presynaptic terminals) which exist at great distances from protein synthetic machinery. To overcome this logistical problem, neurons carry out large amounts of organelle motility along microtubules in the axon. Directed motility along microtubules is generated by molecular motors such as kinesin and cytoplasmic dynein. To understand the mechanism by which motors associate with organelles, subcellular fractions from bovine brain were isolated and characterized with respect to kinesin content by immunochemical methods and electron microscopic immunolocalization. Four subcellular fractions (synaptic vesicles, mitochondria, coated vesicles, and a microsomal fraction) contained detectable levels of kinesin. Immunoelectron microscopy suggested that kinesin was associated with membranes in these fractions and that kinesin was clustered on the surface of mitochondria. These results were consistent with the proposed role of kinesin as a molecular motor for organelle motility. One current model of organelle motility proposes that kinesin drives anterograde organelle motility (away from the cell body) while cytoplasmic dynein drives retrograde motility. This model was tested in a second set of experiments which examined the nucleotide specificity of molecular motors driving organelle motility in squid axoplasm. The nucleotide specificities of anterograde and retrograde organelle motility in this system were indistinguishable implying that molecular motors driving organelle motility in the two directions were enzymatically similar. Given the differing enzymatic characteristics of kinesin and cytoplasmic dynein in vitro, these findings were not consistent with the kinesin/cytoplasmic dynein model of organelle motility as stated above.

A final set of experiments addressed the possible existence of proteinaceous directionality determinants on organelle surfaces. Purified synaptic vesicles were fluorescently labelled and micro-injected into the squid giant synapse. The motile characteristics of synaptic vesicles were altered when synaptic vesicles were proteolyzed prior to micro-injection. In particular, proteolyzed vesicles showed a preference for retrograde motility. These results indicated that organelle directionality is determined, in part, by proteins on the organelle surface.

#### Steve O'Shea

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Rapid membrane repair in two transected giant axons
by Krause, Todd Lawrence, Ph.D., The University of Texas at Austin, 1993, 209 pages; AAT 9323456
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School: The University of Texas at Austin
School Location: United States -- Texas
Index terms(keywords): Loligo pealei, Sepioteuthis lessoniana, Lumbricus terrestris
Source: DAI-B 54/04, p. 1822, Oct 1993
Source type: DISSERTATION
Subjects: Neurology, Cellular biology, Anatomy & physiology, Animals
Publication Number: AAT 9323456
Document URL: http://proquest.umi.com.ezproxy.aut...1&sid=1&Fmt=2&clientId=7961&RQT=309&VName=PQD
ProQuest document ID: 747462731

Abstract (Document Summary)

For a nerve cell to survive transection, toxic changes in intracellular concentrations of ions or macromolecules must be prevented by the formation of a barrier (seal) at the site of damage. Despite the importance of sealing to the survival of damaged neurons and other cell types, sealing has not been well characterized and its mechanism is unknown in any cell type.

Using several functional (membrane potential, input resistance, complex input-impedance, and injury current density) and morphological (phase contrast microscopy, video-enhanced differential interference contrast microscopy, light microscopy and electron microscopy) measures, I assessed sealing in two invertebrate giant axons--the giant axon of squid (Loligo pealei and Sepioteuthis lessoniana) and the medial giant axon of earthworm (Lumbricus terrestris). The functional and morphological data together strongly suggest that when transected in standard salines, the squid axon does not seal within 2.5 hr, whereas the earthworm axon seals within 1 hr. Neither axon seals within 2.5 hr when transected in divalent cation free saline. Further, my data do not support the conventional notion that sealing occurs by constriction and fusion of axolemmal membranes at the cut end. Rather, my data indicates that earthworm axon seals by forming a plug of large $(\ge$5 $\mu$m) injury-induced vesicles at the cut axonal end.

I also assessed the ability of naturally occurring mechanisms of seal formation to facilitate the reconnection of lesioned axons by artificial means (i.e. polyethylene glycol). When the natural ability of earthworm axon to seal-off its cut end was controlled by adjusting the ionic concentration of the bath saline, lesioned axons were efficiently reconnected using polyethylene glycol. The extent of axonal reconnection was extensively characterized using morphological (light microscopy, electron microscopy, passive diffusion of intraxonally injected dye across the reconnection site) and functional (membrane potential, action potential and electrotonic potential conduction through the reconnection site) measures.
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Functional characterization of Electrophorus electrocyte sodium channels
by Shenkel, Scott, Ph.D., Yale University, 1992, 147 pages; AAT 9235556
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School: Yale University
School Location: United States -- Connecticut
Index terms(keywords): Electrophorus electricus
Source: DAI-B 53/07, p. 3350, Jan 1993
Source type: DISSERTATION
Subjects: Anatomy & physiology, Animals, Neurology, Biophysics
Publication Number: AAT 9235556
Document URL: http://proquest.umi.com.ezproxy.aut...1&sid=1&Fmt=2&clientId=7961&RQT=309&VName=PQD
ProQuest document ID: 744439081

Abstract (Document Summary)

The first voltage gated Na channel to be purified biochemically and to be cloned and sequenced is that present in the electrocytes of the electric organ of the electric eel Electrophorus electricus. To complement what is known about its structure my work has focused on the study of the functional properties of the electrocyte Na channel based on electrophysiological measurements of purified, modified reconstituted channels in planar lipid bilayers and unmodified channels in patches of intact electrocyte membrane.

Na channels modified by mild trypsin-treatment and then incorporated into planar lipid bilayers have properties of steady-state voltage-dependence of activation, single-channel conductance, and sensitivity to tetrodotoxin and saxitoxin similar to those of unmodified Na channels in nerve and muscle cell membranes. One exception is the unusually high P$\sb{\rm Na}$/P$\sb{\rm K}$ selectivity ratio of 41, which is two to four times the values previously reported for Na channels in nerve and muscle cells.

My patch-clamp experiments on intact electrocytes have shown that the high P$\sb{\rm Na}$/P$\sb{\rm K}$ observed in the trypsinized channels in bilayers is a real property of the channels in the intact cell membrane. In addition, the trypsinized channels in bilayers and native channels have similar properties of steady-state voltage-dependence of activation and single-channel conductance. Furthermore, the properties of activation, inactivation and single-channel conductance of the electrocyte Na channel are similar to those of Na channels in nerve and muscle cell membranes. A surprising finding is the large variation in P$\sb{\rm Na}$/P$\sb{\rm K}$ among patches from the electrocytes observed under identical recording conditions. My results support the conclusion that there is heterogeneity in the selectivty properties of the Na channels.

The high Na channel density in the intact electrocyte has allowed the recording of gating currents in isolated membrane patches. The properties of these gating currents were found to be similar to those of the squid giant axon and other preparations.

#### Steve O'Shea

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FEEDING STUDIES OF THE MURICID GASTROPOD OCENEBRA ERINACEA (LINNAEUS, 1758), WITH ULTRASTRUCTURAL AND HISTOCHEMICAL INVESTIGATIONS OF THE ABO (ACCESSORY BORING ORGAN)
by HUMPHREY, ELAINE CHRISTINA, Ph.D., University of Southampton (United Kingdom), 1990, 185 pages; AAT DX94706
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School: University of Southampton (United Kingdom)
School Location: England
Source: DAI-B 52/10, p. 5121, Apr 1992
Source type: DISSERTATION
Subjects: Oceanography
Publication Number: AAT DX94706
Document URL: http://proquest.umi.com.ezproxy.aut...1&sid=1&Fmt=2&clientId=7961&RQT=309&VName=PQD
ProQuest document ID: 745393841

Abstract (Document Summary)

Available from UMI in association with The British Library.

The response to prey and the general feeding behaviour of Ocenebra erinacea was observed together with feeding rates under specific laboratory conditions of different light, temperature and salinity regimes.

The ultrastructure of the accessory boring organ (ABO) was investigated by standard electron microscopical techniques and development of the ODO method gaining a spacial interpretation of this structurally complex organ. In the full description of the ABO, septate junctions in the apical region of the ABO, and "beaded" microvilli, were recorded.

A number of histochemical studies, mostly at the ultrastructural level, were undertaken including Ca-ATPase, Na-K-ATPase, acid phosphatase, adenylate cyclase, substance P, serotonin, a tracer study using horseradish peroxide and an etching study of the secretion of the ABO on the chitin/protein complex of squid pen, furthering information chronicled on the muricid ABO. The enzyme results indicated at least three domains in the plasmalemma of the epithelial cells: the apical membrane, the contralacunal membrane, and the intercolumnar membrane.

Whilst investigating the developing ABO in the larvae, a method for inducing egg capsule production in the laboratory was formulated.
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Canales ionicos de neuronas septales aisladas: Caracterizacion electrofisiologica y modulacion por TRH y LHRH
by Castellano Orozco, Antonio Gonzalo, xx, Universidad de Sevilla (Spain), 1989, 128 pages; AAT C145815
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Alternate Language Title: Ionic channels in septal neurons: Characterization and modulation by TRH and LHRH
Language: Spanish , ES
School Location: Spain
Source: DAI-C 51/04, p. 541, Winter 1990
Source type: DISSERTATION
Subjects: Anatomy & physiology, Animals
Publication Number: AAT C145815
ISBN: 8474054826
Document URL: http://proquest.umi.com.ezproxy.aut...1&sid=1&Fmt=2&clientId=7961&RQT=309&VName=PQD
ProQuest document ID: 743969711

Abstract (Document Summary)

The ionic currents of dispersed septal neurons were studied using the "whole-cell" variant of the "patch-clamp" technique. On depolarization all septal neurons generate Na, Ca, and K currents. The Na current activates rapidly and deactivates in 3 to 4 ms. This current is completely blocked by TTX and has characteristics similar to the ones of the Na current described by Hodgkin and Huxley in the squid giant axon. The calcium current is due to the activity of two types of calcium channels with properties similar to the fast (FD) and slow (SD) deactivating channels of other cells. The potassium current is mediated by at least three different K channels. There is a fast transient current which is deactivated at potentials more positive than $-$50 mV. At +40 mV this current deactivates almost totally in 20 ms. In addition, there are two components of slowly activating outward current, one of them with a clear dependence on the intracellular Ca$\sp{2+}$ concentration. The density of Na channels and the contribution of the different Ca and K channels to the total current varied from cell to cell. This indicates that there are several neuronal types in the septal nucleus. In some cells exposure to external TRH (1 $\mu$M) produces a reversible increase in the amplitude of the slow activating potassium current. In other cells, exposure to 1-3 $\mu$M TRH$\sb{\rm a}$ (acid form) produces a reversible decrease of the Na current amplitude without affecting either the activation and deactivation time courses or the shape of the I-V curve. A second exposure to TRH$\sb{\rm a}$ produces a lower effect. These results suggest the existence of a TRH receptor in the membrane of some cells of the septal nucleus. In some of the cells in which TRH$\sb{\rm a}$ previously produced a decrease in Na current amplitude, the exposure to 2-3 $\mu$M LHRH produces the same effect as the second exposure to TRH$\sb{\rm a}$.

#### Steve O'Shea

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STRUCTURE AND BIOCHEMISTRY OF SQUID PHOTORECEPTOR MICROVILLI
by LANGMACK, KEITH ANTONY, D.Phil., University of Oxford (United Kingdom), 1989, 170 pages; AAT D-91445
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School: University of Oxford (United Kingdom)
School Location: England
Source: DAI-B 51/10, p. 4737, Apr 1991
Source type: DISSERTATION
Subjects: Zoology, Oceanography, Anatomy & physiology, Animals, Biochemistry, Ophthalmology
Publication Number: AAT D-91445
Document URL: http://proquest.umi.com.ezproxy.aut...1&sid=1&Fmt=2&clientId=7961&RQT=309&VName=PQD
ProQuest document ID: 745062521

Abstract (Document Summary)

Available from UMI in association with The British Library. Requires signed TDF.

This thesis reports results of studies on the proteins of squid photoreceptor microvilli. This serves as a model system both for signal transduction and for membrane-cytoskeletal interactions, which are crucial processes in the response of cells to external stimuli. The approach used here has been both biochemical and structural. First of all, the glycosylation of the microvillar proteins was investigated by lectin affinity blotting. This revealed that some of the cytoskeletal proteins were glycosylated. This gave a possible explanation for the cytoskeletal aggregating on isolation with non-ionic detergent. Some of the cytoskeletal proteins are transmembrane and the hydrophobic regions, exposed on addition of detergent, associate in a non-specific manner to produce the aggregation. The aggregates were reduced in size by the addition of high concentrations of salt. It was found that salt-solubilised proteins re-aggregated on the addition of micromolar quantities of Mg-ATP. This also results in the phosphorylation of two of the cytoskeletal proteins (molecular weights 110 and 130 kDa). A cytoskeletal protein of 110 kDa binds actin and cross-reacts with an antimyosin head monoclonal antibody. This protein was therefore thought to be a myosin I. Negative stain electron microscopy of the cytoskeleton has also shown the aggregation.

The structure of the microvillar membranes was studied by negative staining. This revealed projections from the surface that were associated with membrane-membrane interactions. By labelling the membranes with concanavalin A-gold, it was shown that these projections were most likely to be on the cytoplasmic surface of the membranes. Rhodopsin was purified by chromatography and reconstituted into lipid vesicles. These vesicles showed similar projections to the native membranes, indicating that the projections contain rhodopsin. Rhodopsin in native membranes was observed in partially ordered arrays.

The G$\sb{\beta}$ subunit (the $\beta$ subunit of a GTP-binding protein) was isolated by sucrose density gradient centrifugation. Vesicles containing this polypeptide had a morphology that was dependent on their lipid content rather than their protein content.
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INVESTIGATION OF THE MOLECULAR BASIS OF DIRECTED PARTICLE MOVEMENT IN THE SQUID GIANT AXON (VESICLES, CELL MOTILITY, FAST AXONAL TRANSPORT)
by GILBERT, SUSAN POND, Ph.D., Dartmouth College, 1986, 121 pages; AAT 8628285
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School: Dartmouth College
School Location: United States -- New Hampshire
Source: DAI-B 47/09, p. 3615, Mar 1987
Source type: DISSERTATION
Subjects: Biology
Publication Number: AAT 8628285
Document URL: http://proquest.umi.com.ezproxy.aut...1&sid=1&Fmt=2&clientId=7961&RQT=309&VName=PQD
ProQuest document ID: 751453941

Abstract (Document Summary)

The research presented in this thesis was designed to provide new information about the motive force generating system responsible for the important neurological process, fast axonal transport. This process involves the rapid, bidirectional movement of membranous organelles along the length of the axon and is responsible for transfer of proteins and neurotransmitters or their precursors to the synapse. To learn more about this process, a model system was developed using isolated axoplasmic vesicles from the squid giant axon. The membranes of the vesicles were labeled fluorescently and were made to move either by injecting them into another axon or by allowing them to associate with an oriented microtubule substrate in the form of flagellar axonemal microtubules. In both model systems, the exogenous vesicles were observed to move bidirectionally and in an ATP dependent and trypsin sensitive manner. Furthermore, these vesicles were observed to translocate on isolated axonemal microtubules in the presence or absence of flagellar dynein. Because the vesicles were observed to move in either direction on a single axoneme, the inherent structural polarity of the microtubule did not specify the direction of vesicle movement. Moreover, the vesicle motility was ATP dependent and trypsin sensitive, yet flagellar dynein was not responsible for the vesicle translocations.

Because isolated microtubules were shown to support vesicle motility, it was assumed that the ATP binding proteins essential for vesicle translocation were present in the vesicle preparation. To identify these molecules, the radioactive photoaffinity analog of ATP, (alpha)('32)P 8-azidoadenosine 5'-triphosphate, was used. The results identify and characterize a vesicle associated polypeptide that bound the analog. This polypeptide was isolated in association with vesicles or stabilized microtubules and migrated at an M(,r) of 292 kd. Furthermore, the 292 kd polypeptide crossreacted with antiserum to porcine brain microtubule associated protein 2 (MAP 2). Because the 292 kd polypeptide binds ATP, is associated with axoplasmic vesicles that translocate on MAP-free microtubules in an ATP dependent manner, and can be isolated in association with vesicles and microtubules, this MAP 2-like polypeptide is believed to be involved in vesicle-microtubule interactions that promote vesicle motility.

#### Steve O'Shea

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TRANSPORT OF SECRETORY VESICLES IN MURINE ANTERIOR PITUITARY TUMOR CELLS (FAST, REGULATED SECRETION, ATT-20 CELLS)
by SCHROER, TRINA ALEXIS, Ph.D., University of California, San Francisco, 1986, 157 pages; AAT 8613054
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School: University of California, San Francisco
School Location: United States -- California
Source: DAI-B 47/03, p. 1044, Sep 1986
Source type: DISSERTATION
Subjects: Biochemistry
Publication Number: AAT 8613054
Document URL: http://proquest.umi.com.ezproxy.aut...1&sid=1&Fmt=2&clientId=7961&RQT=309&VName=PQD
ProQuest document ID: 753313461

Abstract (Document Summary)

Secretory and membrane proteins destined for export from the cell move to the cell surface in membrane vesicles. Two biochemically distinct classes of secretory vesicles exist in the murine anterior pituitary tumor cell line AtT-20. I have demonstrated using indirect immunofluorescence that proteins carried in the different vesicles have different intracellular destinations. ACTH is concentrated at the tips of cell processes and in the Golgi region. In contrast, the constitutively exported murine leukemia virus membrane glycoprotein is observed only in the cell body plasma membrane and not in processes. The influenza virus membrane glycoprotein, hemagglutinin, introduced into cells by transfection, is also constitutively exported and is found exclusively on the cell body surface. Constitutive secretory vesicles appear to release their contents only at the surface of the cell body and not in processes.

Secretion from the regulated and constitutive secretory pathways is inhibited to different degrees by reduced temperature. Constitutive secretion of ACTH precursor and the secreted form of vesicular stomatitis virus glycoprotein is inhibited approximately 7-fold at 20(DEGREES)C. In the regulated pathway, ACTH precursor is processed to mature ACTH and stored in dense core secretory granules at least as well at 20(DEGREES)C as at 37(DEGREES)C. Autoradiography of sulfate labeled cells indicated that secretory granules are transported normally to the ends of processes at 20(DEGREES)C. However, regulated secretion of mature ACTH is inhibited completely.

The movement of isolated secretory vesicles along axonal microtubules was examined in vitro. Purified synaptic vesicles from electric rays were fluorescently labeled and their movement in cytoplasm isolated from squid giant axons was examined by fluorescence microscopy. The synaptic vescles behaved like endogenous squid organelles, moving preferentially toward the synapse at a velocity of 2 microns per second and binding to microtubules when movement was inhibited by a non-hydrolyzable ATP analog. Vesicles that had been proteolytically digested with trypsin or pronase were observed to move in axoplasm and to bind to microtubules but the direction preference was abolished. Vesicles treated with elastase neither moved nor bound. Apparently, different synaptic vesicle membrane proteins are responsible for the property of microtubule binding and the preference for anterograde movement.
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STUDIES OF TWO NATURALLY OCCURRING COMPOUNDS WHICH EFFECT RELEASE OF ACETYLCHOLINE FROM SYNAPTOSOMES
by KOENIG, MICHAEL LEO, Ph.D., University of Southern California, 1985; AAT 0557728
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School: University of Southern California
School Location: United States -- California
Source: DAI-B 46/12, p. 4098, Jun 1986
Source type: DISSERTATION
Subjects: Biology
Publication Number: AAT 0557728
Document URL: http://proquest.umi.com.ezproxy.aut...1&sid=1&Fmt=2&clientId=7961&RQT=309&VName=PQD
ProQuest document ID: 752246591

Abstract (Document Summary)

Two naturally occurring compounds which effect the release of neurotransmitter from synaptosomes have been purified to apparent homogeneity. Iotrochotin (IOT) isolated from wound exudate of the Caribbean purple bleeder sponge promotes release in a manner that is independent of the extracellular Ca('2+) ion concentration. Leptinotarsin (LPT-d), a protein taken from hemolymph of the Colorado potato beetle, Leptinotarsa decemlineata, stimulates Ca('2+)-dependent release.

IOT, purified by conventional chromatographic techniques, migrates as a single band on sodium dodecylsufate polyacrylamide gel electrophoresis (SDS-PAGE) slab gels. The protein is slightly acidic and has a molecular weight of approximately 18 kD. ('3)H Acetylcholine which has been introduced into synaptosomes as ('3)H choline can be released by IOT. The toxin releasable pool of labelled neurotransmitter is not depleted by depolarization of the synaptosomes with high potassium, and therefore seems to be primarily extravesicular. That IOT has membrane permeabilizing properties is suggested by the finding that small cytoplasmic molecules can be co-released with neurotransmitter. Adenosine triphosphate and exogenously added 2- ('3)H deoxy-D-glucose are readily released by the toxin, whereas larger cytoplasmic marker enzymes are not.

LPT-d is a larger protein (molecular weight = 45 kD) than IOT, and seems to effect primarily vesicular release by opening at least one type of presynaptic Ca('2+) channel. The facilitatory effects of the toxin on synaptosomal release can be inhibited by inorganic Ca('2+) channel antagonists, but are not generally affected by organic antagonists thought to be most effective as inhibitors of slow Ca('2+) channels. There is some evidence that the organic antagonist nifedipine, if added after LPT-d, can close the toxin-activated channels in cultured rat diencephalon cells. If so, the channels affected may be homologous with those found in smooth muscle and heart. Studies of the effects of LPT-d on Ca('2+) channels from a variety of tissues including Paramecium, squid optic lobe, smooth and skeletal muscle, and neuronal clonal cell lines, have shown the toxin to be an activator specifically of a type of channel found only in the mammalian central nervous system.

(Copies available exclusively from Micrographics Department, Doheny Library, USC, Los Angeles, CA 90089-0182.)

#### Steve O'Shea

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DIELECTRIC PROPERTIES OF THE SQUID GIANT AXON MEMBRANE
by FERNANDEZ, JULIO MARIANO, Ph.D., University of California, Los Angeles, 1982, 81 pages; AAT 8219681
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School: University of California, Los Angeles
School Location: United States -- California
Source: DAI-B 43/04, p. 962, Oct 1982
Source type: DISSERTATION
Subjects: Biophysics
Publication Number: AAT 8219681
Document URL: http://proquest.umi.com.ezproxy.aut...1&sid=1&Fmt=2&clientId=7961&RQT=309&VName=PQD
ProQuest document ID: 753149681

Abstract (Document Summary)

Dielectric polarization of the cell membrane resulting from distortion and alignment of molecules under the influence of the applied electric field has been demonstrated in the squid giant axon membrane.

A method of measuring membrane dielectric polarization is developed in this dissertation. The method consists of measuring the complex admittance of the squid giant axon membrane under voltage clamp in the absence of ionic conductances for different membrane potentials. This is accomplished by adding a small Pseudo Random Binary Sequence (PRBS) to the membrane potential and the admittance is obtained from the applied membrane potential and its current response using Fourier techniques. Detailed design specifications are given for the implementation of the PRBS noise generator.

The results show that the membrane admittance is voltage dependent in the range between -120 mV and 50 mV and it is independent of the membrane potential outside this range. The voltage dependent part of the admittance can be related to the movement of charges responsible for the opening and closing of the sodium channel demonstrated previously by other techniques (Armstrong and Bezanilla, 1973).

The voltage independent admittance shows characteristics of dielectric loss which can be related to the movement of charges or dipoles which are likely to be associated to those intrinsic proteins of the membrane whose function is other than the gating of voltage dependent ionic channels.

The admittance results support the view that the membrane is a mosaic of regions with different dielectric properties. This hypothesis is supported by experiments showing that the movement of charges responsible for sodium channel gating respond differently than lipophilic ions to perturbations of the lipid matrix. This suggests that the gating charge movement occurs in a region order than the bulk lipid matrix.
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CALCIUM HOMEOSTASIS AND THE EFFECT OF ANTICHOLINERGIC COMPOUNDS IN RAT BRAIN
by MELAMED, BRIAN RICHARD, Ph.D., Temple University, 1980, 112 pages; AAT 8025145
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School: Temple University
School Location: United States -- Pennsylvania
Source: DAI-B 41/05, p. 1736, Nov 1980
Source type: DISSERTATION
Subjects: Pharmacology
Publication Number: AAT 8025145
Document URL: http://proquest.umi.com.ezproxy.aut...1&sid=1&Fmt=2&clientId=7961&RQT=309&VName=PQD
ProQuest document ID: 753778231

Abstract (Document Summary)

The influence of QNB (3-quinuclidinyl benzilate), benactyzine, atropine, scopolamine, dexetimide (d-benzetimide), and levetimide (l-benzetimide) on the calcium content of rat brain cortex slices was investigated. Slices were incubated either in "normal" (5 mMCl) glucose-bicarbonate medium at 37(DEGREES) for 60 min. or were depolarized by incubation in a medium containing 100 mM KCl. It was found that all of the compounds tested exerted effects only on depolarized slices. QNB at 0.1 and 1.0 mM inhibited the increase in calcium content of the depolarized slices. Studies of calcium efflux from ('45)Ca preloaded slices indicated that low concentrations of QNB (0.1, 0.01 mM) inhibited calcium extrusion while at high concentration (1.0 mM) QNB produced a slight enhancement of the rate of calcium efflux. Similar effects on calcium content were noted for benactyzine which was equipotent to QNB except at the 10 (mu)M concentration where no effect was observed. The stereoisomers, d- and l-benzetimide were approximately equipotent in their effects on calcium although the d-isomer has been shown to have 2000 times greater affinity for the muscarinic receptor. This would indicate that the calcium antagonistic effect is not mediated via an action on the muscarinic receptor. The belladonna alkaloids, atropine and scopolamine were considerably less potent than QNB. When the "lanthanum technique" was employed to displace extracellularly bound Ca('++) and to measure intracellular Ca('++) levels, it was found that 1, 10, and 100 (mu)M QNB all produced approximately the same degree of inhibition of K('+) - stimulated calcium accumulation. The effect of QNB on subcellular calcium storage sites showed that concentrations above 0.1 mM were required to inhibit energy-dependent calcium accumulation by rat brain mitochondrial and microsomal fractions. The effects were achieved in a calmodulin-independent manner, indicating therefore, that calmodulin was probably not a primary site of action. Studies in slices with vanadate ion, a specific inhibitor of ATP-dependent sodium independent, calcium pumping mechanisms in squid axons, produced an increase in calcium content that produced by omission of Na('+) ions. This observation provides further evidence for the existence of a dual mechanism for the extrusion of calcium from mammalian nervous tissue. Under conditions in which both efflux mechanisms were inhibited, QNB decreased the calcium content relative to the inhibited slices. The level of calcium achieved was still greater than that of the control slices. This indicated that blockade of calcium channels by QNB was at best incomplete and inadequate to explain the large decrease in calcium content observed. It was concluded that QNB displaced calcium from binding sites within the cell. The site of intracellular calcium storage affected was most likely the inner aspect of the plasma membrane. Some blockade of calcium channels must also occur in light of the observation that QNB decreased calcium content while inhibiting efflux. Results are discussed with regard to the overall nature and mechanisms of calcium storage in brain tissue. In addition, a suggestion is made relating the increased ability of QNB to alter calcium metabolism and the greater psychotomimetic potency of some of the glycolate esters as compared to other classes of anticholinergic compounds.

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