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Biomedical subjects

P B Guthrie

Publications and source records attributed to P B Guthrie.

At least 19 recordsLinked to original sources
PubMed2000

Green fluorescent protein as a quantitative tool.

Manipulating the expression of a protein can provide a powerful tool for understanding its function, provided that the protein is expressed at physiologically-significant concentrations. We have developed a simple method to measure (1) the concentration of an overexpressed protein in single cells and (2) the covariation of particular physiological properties with a protein's expression. As an example of how this method can be used, teratocarcinoma cells were transfected with the neuron-specific calcium binding protein calretinin (CR) tagged with green fluorescent protein (GFP). By measuring GFP fluorescence in microcapillaries, we created a standard curve for GFP fluorescence that permitted quantification of CR concentrations in individual cells. Fura-2 measurements in the same cells showed a strong positive correlation between CR-GFP fusion protein expression levels and calcium clearance capacity. This method should allow reliable quantitative analysis of GFP fusion protein expression.

Animals↗
PubMed2000

Stimulus history alters behavioral responses of neuronal growth cones.

Generally, it is assumed that growth cones respond to a specific guidance cue with a single, specific, and stereotyped behavior. However, there is evidence to suggest that previous exposure to a given cue might alter subsequent responses to that cue (Snow and Letourneau, 1992; Shirasaki et al., 1998). We therefore tested the hypothesis that growth cone responses to stimuli are dependent on the history of previous stimulation. Growth cones of chick dorsal root ganglion neurons were exposed to well characterized stimuli: (1) contact with a laminin-coated bead, which causes growth cone turning, or (2) electrical stimulation, which causes growth cone collapse. Although the expected behavioral responses were observed after the initial stimulation, strikingly different responses to a subsequent stimulation were observed. Growth cones that had recovered from electrical stimulation-induced collapse rapidly developed insensitivity to a second identical electrical stimulation. Growth cones that previously turned in response to contact with a laminin-coated bead responded to a second bead with a "stall" or cessation in outgrowth. This stimulus history dependence of growth cone behavior could be generalized across dissimilar stimuli: after contact with a laminin-coated bead, growth cones failed to collapse in response to electrical stimulation. The calcium/calmodulin-dependent protein kinase II (CaMKII) was implicated in this history dependence by pharmacological experiments. Together, these results demonstrate that growth cones can alter their behavioral response rapidly to a given stimulus in a manner dependent on previous history and that knowledge of past events in growth cone navigation may be required to predict future growth cone behavior.

Animals↗
PubMed1999

Membrane recycling in the neuronal growth cone revealed by FM1-43 labeling.

Membrane dynamics within the chick ciliary neuronal growth cone were investigated by using the membrane-impermeant dye FM1-43. A depolarization-evoked endocytosis was observed that shared many properties with the synaptic vesicle recycling previously described at the presynaptic terminal. In addition, in the absence of depolarization a basal level of constitutive endocytotic activity was observed at approximately 30% of the rate of evoked endocytosis. This constitutive endocytosis accounted for large amounts of membrane retrieval: the equivalent of the entire growth cone surface area could be internalized within a 30 min period. Endosomes generated via constitutive and evoked processes were highly mobile and could move considerable distances both within the growth cone and out to the neurite. In addition to their different requirements for formation, evoked and constitutive endosomes displayed a significant difference in release properties. After a subsequent depolarization of labeled growth cones, evoked endosomes were released although constitutive endosomes were not released. Furthermore, treatment with latrotoxin released evoked endosomes, but not constitutive endosomes. Although the properties of evoked endosomes are highly reminiscent of synaptic vesicles, constitutive endosomes appear to be a separate pool resulting from a distinct and highly active process within the neuronal growth cone.

Animals↗
PubMed1999

ATP released from astrocytes mediates glial calcium waves.

Calcium waves represent a widespread form of intercellular communication. Although they have been thought for a long time to require gap junctions, we recently demonstrated that mouse cortical astrocytes use an extracellular messenger for calcium wave propagation. The present experiments identify ATP as a major extracellular messenger in this system. Medium collected from astrocyte cultures during (but not before) calcium wave stimulation contains ATP. The excitatory effects of medium samples and of ATP are blocked by purinergic receptor antagonists and by pretreatment with apyrase; these same purinergic receptor antagonists block propagation of electrically evoked calcium waves. ATP, applied at the concentration measured in medium samples, evokes responses that are qualitatively and quantitatively similar to those evoked by those medium samples. These data implicate ATP as an important transmitter between CNS astrocytes.

Adenosine Triphosphate↗
PubMed1998

An overview of aesthetic surgery of the face.

This article explores the interface between the aesthetic aspects of dentistry and complementary cosmetic surgery of the face. Dentists do not often think of facial cosmetic surgery and plastic surgeons do not often think of aesthetic dentistry as being complementary to their endeavours. This paper is designed to help breach this gap. An outline is given of the various cosmetic surgery procedures used on the face with emphasis on those in the perioral region. Procedures discussed include meloplasty, blepharoplasty, laser resurfacing, liposculpture, lip augmentation, rhinoplasty, chin augmentation, correction of prominent ears, and skin care.

Esthetics, Dental↗
PubMed1996

An extracellular signaling component in propagation of astrocytic calcium waves.

Focally evoked calcium waves in astrocyte cultures have been thought to propagate by gap-junction-mediated intercellular passage of chemical signal(s). In contrast to this mechanism we observed isolated astrocytes, which had no physical contact with other astrocytes in the culture, participating in a calcium wave. This observation requires an extracellular route of astrocyte signaling. To directly test for extracellular signaling we made cell-free lanes 10-300 microns wide in confluent cultures by deleting astrocytes with a glass pipette. After 4-8 hr of recovery, regions of confluent astrocytes separated by lanes devoid of cells were easily located. Electrical stimulation was used to initiate calcium waves. Waves crossed narrow (< 120 microns) cell-free lanes in 15 of 36 cases, but failed to cross lanes wider than 120 microns in eight of eight cases. The probability of crossing narrow lanes was not correlated with the distance from the stimulation site, suggesting that cells along the path of the calcium wave release the extracellular messenger(s). Calculated velocity across the acellular lanes was not significantly different from velocity through regions of confluent astrocytes. Focal superfusion altered both the extent and the direction of calcium waves in confluent regions. These data indicate that extracellular signals may play a role in astrocyte-astrocyte communication in situ.

Animals↗
PubMed1996

Spatial gradients of cytosolic calcium concentration in neurones during paradoxical activation by calcium.

4-Br-A23187 caused a calcium influx into chick sensory neurones and raised cytosolic calcium from a rest level of 97 +/- 7 nM to a peak of 296 +/- 30 nM. Despite the continued presence of ionophore, however, cytosolic calcium concentrations then fell. After 30 min in ionophore, cytosolic calcium concentration had returned to 105 +/- 5 nM, not significantly different from the value before ionophore addition. The permeability of the plasmalemma to divalent cations, as estimated by the manganese quench technique, was no lower at 30 min than at the peak of the cytosolic calcium transient. Thus the fall of calcium from its peak was not due to a slowing of calcium influx, but was due to an upregulation of mechanisms that remove calcium from the cytosol- an upregulation that persists even though cytosolic calcium has apparently returned to pre-stimulus levels. We used a novel fixed slit confocal microscope to examine the calcium concentration profile close to the plasmalemma. We found that after 25-30 min ionophore treatment, calcium concentration was elevated only in the cytoplasm within 1 micron of the plasmalemma. A maintained, elevated calcium under the plasmalemma can help explain the phenomenon of paradoxical activation seen in this and other cell types.

Animals↗
PubMed1996

Malignant lesions of the lips.

As for all cancers successful treatment of carcinoma of the lip depends on early detection and treatment. This article describes the different types of carcinoma of the lip with notes on their aetiology, diagnosis, natural histories, treatment and prevention. The observation is made that maxillofacial surgeons are well placed to make an early diagnosis which leads to decreased morbidity and mortality.

Basal Cell Carcinoma↗
PubMed1994

Self-recognition: a constraint on the formation of electrical coupling in neurons.

Electrical coupling between specific neurons is important for proper function of many neuronal circuits. Identified cultured neurons from the snail Helisoma show a strong correlation between electrical coupling and presence of gap junction plaques in freeze-fracture replicas. Gap junction plaques, however, were never seen between overlapping neurites from a single neuron, even though those same neurites formed gap junctions with neurites from another essentially identical identified neuron. This observation suggests that a form of self-recognition inhibits reflexive gap junction formation between sibling neurites. When one or both of those growth cones had been physically isolated from the neuronal cell body, both electrical coupling and gap junction plaques, between growth cones from the same neuron, were observed to form rapidly (within 30 min). Thus, inhibition of electrical coupling between sibling neurites apparently depends on cytoplasmic continuity between neurites, and not the molecular composition of neurite membrane. The formation of gap junctions is not likely due to the isolation process; rather, the physical isolation appears to release an inhibition of reflexive gap junction formation. These data demonstrate the existence of a previously unknown constraint on the formation of electrical synapses.

Animals↗
PubMed1993

Accommodation of mouse DRG growth cones to electrically induced collapse: kinetic analysis of calcium transients and set-point theory.

Electrical stimulation causes growth cones of mouse dorsal root ganglion neurons to collapse. During chronic stimulation, however, growth cones resume motility. In addition, these growth cones are now resistant to the collapsing effects of subsequent stimulation, a process we term accommodation. We compared the kinetics of electrically induced Ca2+ transients in naive and accommodated growth cones in order to determine whether the accommodation process results from a change in the Ca2+ transient, or a change in the Ca2+ sensitivity of the growth cones. Three kinetics were determined: (1) the initial increase to peak Ca2+ levels produced by 10 Hz stimulation; (2) recovery from peak Ca2+ levels during stimulus trains lasting 15 min; and (3) clearing of Ca2+ from growth cones after terminating the stimulus. These kinetics were analyzed using single exponential fits to changes in fura-2 fluorescence ratios. The electrically evoked increase in Ca2+ was significantly slower in accommodated growth cones (tau = 6.0 s) compared to naive growth cones (tau = 1.4 s). Despite the slower increase of [Ca2+]i in accommodated growth cones, peak [Ca2+]i was similar to that reached in naive growth cones, and the steady-state Ca2+ level was significantly elevated after chronic stimulation. Thus, accommodated growth cones maintained outgrowth at [Ca2+]i that caused collapse initially. Time course experiments show that accommodation is a slow process (t 1/2 = about 3 h). Accommodation did not induce measurable changes in the rates of Ca2+ homeostasis during or after stimulus trains. The kinetics of Ca2+ recovery during (tau = 90 s) and after 15 min of stimulation (tau = 8.5 s) was not significantly different in accommodated versus naive growth cones. Rates of 45Ca2+ efflux were also similar in both types of growth cones. These results suggest two regulatory processes contributing to growth cone motility during chronic stimulation: (1) recovery of [Ca2+]i to levels permissive to neurite outgrowth, and (2) an increase in the range of optimal [Ca2+]i for growth cone motility. These adaptive responses of mammalian growth cones to chronic stimulation could be involved in the modulation of CNS development by electrical activity of neurons.

Adaptation, Physiological↗
PubMed1993

Local calcium regulatory compartments in neurons.

We have demonstrated that calcium concentration is under local control in two different systems: hippocampal pyramidal cell dendritic spines, and developing grasshopper pioneer neuron axons. Controllable, photoinduced damage to fura-2 filled processes was used for generating defined, reliable cytoplasmic calcium transients to demonstrate these local calcium regulatory compartments.

Animals↗
PubMed1991

Independent regulation of calcium revealed by imaging dendritic spines.

The dendritic spine is a basic structural unit of neuronal organization. It is assumed to be a primary locus of synaptic plasticity, and to undergo long-term morphological and functional changes, at least some of which are regulated by intracellular calcium concentrations. It is known that physiological stimuli can cause marked increases in intracellular calcium levels in hippocampal dendritic shafts, but it is completely unknown to what extent such changes in the dendrites would also be seen by calcium-sensing structures within spines. Will calcium levels in all spines change in parallel with the dendrite or will there be a heterogeneous response? This study, through direct visualization and measurement of intracellular calcium concentrations in individual living spines, demonstrates that experimentally evoked changes in calcium concentrations in the dendritic shaft ([Ca2+]d).

Animals↗
PubMed1991

Sustained increase in intracellular calcium promotes neuronal survival.

Ciliary ganglion neurons, half of which normally suffer developmental death in the embryo, will survive in culture in medium supplemented with depolarizing concentrations of potassium. It is not known how elevated potassium acts inside the cell to promote survival. We report here that depolarizing concentrations of extracellular potassium promote neuronal survival by causing a sustained increase in intracellular calcium. Raising extracellular potassium from 5 to 40 mM, an optimal concentration for survival, caused a sustained increase in intracellular calcium from 250 nM to greater than 600 nM. By 26 hr, at which time greater than 90% of neurons in 5 mM potassium had died, the calcium concentration of neurons in 40 mM potassium was still above 400 nM. Reduction of extracellular potassium from 40 to 5 nM, which prevents the increase in survival, also reduced intracellular calcium back to rest levels. PN200-110, a dihydropyridine calcium channel blocker that inhibits the survival-promoting effect of elevated potassium, also prevented and reversed the potassium,-mediated increase in intracellular calcium. In addition, there was a strong, quantitative correlation between the percentage of neuronal survival and the intracellular calcium concentration over a wide range of extracellular potassium concentrations. These results suggest that elevated potassium opens dihydropyridine-sensitive calcium channels, causing a sustained increase in intracellular calcium that quantitatively determines the number of surviving neurons.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1991

Rhinoplasty--the other facial osteotomy.

Orthognathic surgery commenced with mandibular procedures, progressed to maxillary procedures, and then onto bimaxillary procedures. So far, there has been an inadequate appreciation of the need to include nasal appearance in orthognathic planning. This paper discusses the relationship of rhinoplasty to orthognathic surgery, and outlines an operative technique for rhinoplasty.

Humans↗
PubMed1991

Calcium ion distribution in nascent pioneer axons and coupled preaxonogenesis neurons in situ.

The "calcium hypothesis" of regulation of growth cone motility and neurite elongation has derived from analysis of a variety of neurons growing in vitro. It proposes that calcium ion concentration within growth cones is an important regulator of motility and growth. We now extend this analysis by investigating calcium concentrations within growth cones and nascent neurites of identified embryonic neurons growing on their normal substrate in situ. The pair of Ti1 pioneer neurons are the first to extend axons in limb buds of grasshopper embryos. Their growth cones migrate along a stereotyped pathway, where they encounter a series of guidance cues, including preaxonogenesis afferent neurons (guidepost cells). By injecting a pioneer neuron with fura-2 dye, we measured calcium concentration in the injected neuron, and in cells to which it became dye-coupled, at successive stages of pioneer outgrowth through the limb. Pioneer neurons undergoing axonogenesis had calcium concentrations in the above-100-nM range characteristic of a variety of neuron types studied in vitro. In pioneer neurons not yet in contact with guidepost cells, a shallow calcium concentration gradient, highest at the growth cone, was often observed. Guidepost cells that had not begun axonogenesis had lower calcium concentrations, in the 65 nM range. Therefore, an increase in cytosolic calcium concentration may be associated with the onset of axonogenesis. Nascent pioneer neurons were fura-2 dye-coupled to each other and established coupling with contacted guidepost cells. Calcium concentration measurements along pioneer neurites suggest that calcium ions also are transferred from pioneer neurons to these coupled guidepost cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Localized calcium influx orients axon formation in embryonic hippocampal pyramidal neurons.

A fundamental property of neurons is their polarization into distinct axonal and dendritic compartments which have characteristic structural and functional properties. The mechanisms regulating the formation of neuronal polarity are unknown. We used cultured embryonic rat hippocampal pyramidal neurons to test the hypothesis that a localized calcium influx can orient axon formation, and thereby direct the establishment of neuronal polarity. Transection of an initial axon, or focal application of A23187 or K+ to the initial axon, caused a new axon to form at a site distant from the initial axon. Fura-2 measurements of intracellular calcium revealed a localized calcium influx at the site of axon transection or focal application of A23187 or K+, and a calcium gradient spreading into the soma. New axon formation was inhibited when axons were transected in medium lacking calcium or containing calcium-elevating agents (conditions which prevented the formation of a calcium gradient). When calcium ionophore A23187 was applied focally to neurons which had not yet established an axon, the axon always formed at a site distant from the site of ionophore application; bath exposure to A23187 prevented axon formation. Taken together, these data demonstrate that a localized influx of calcium can suppress axon formation at the site of influx, and can thereby influence where the axon forms. These data suggest that gradients of intracellular calcium may be involved in orienting neuronal polarity.

Animals↗