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1.
J Vet Pharmacol Ther ; 38(4): 330-5, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25376083

ABSTRACT

The objective of this study was to determine the disposition of ampicillin in plasma, uterine tissue, lochial fluid, and milk of postpartum dairy cattle. Ampicillin trihydrate was administered by intramuscular (i.m.) injection at a dose of 11 mg/kg of body weight every 24 h (n = 6, total of 3 doses) or every 12 h (n = 6, total of 5 doses) for 3 days. Concentrations of ampicillin were measured in plasma, uterine tissue, lochial fluid, and milk using HPLC with ultraviolet absorption. Quantifiable ampicillin concentrations were found in plasma, milk, and lochial fluid of all cattle within 30 min, 4 h, and 4 h of administration of ampicillin trihydrate, respectively. There was no significant effect of dosing interval (every 12 vs. every 24 h) and no significant interactions between dosing interval and sampling site on the pharmacokinetic variable measured or calculated. Median peak ampicillin concentration at steady-state was significantly higher in lochial fluid (5.27 µg/mL after q 24 h dosing) than other body fluids or tissues and significantly higher in plasma (3.11 µg/mL) compared to milk (0.49 µg/mL) or endometrial tissue (1.55 µg/mL). Ampicillin trihydrate administered once daily by the i.m. route at the label dose of 11 mg/kg of body weight achieves therapeutic concentrations in the milk, lochial fluid, and endometrial tissue of healthy postpartum dairy cattle.


Subject(s)
Ampicillin/pharmacokinetics , Body Fluids/chemistry , Cattle/metabolism , Milk/chemistry , Postpartum Period/physiology , Uterus/metabolism , Ampicillin/blood , Animals , Anti-Bacterial Agents/blood , Anti-Bacterial Agents/pharmacokinetics , Cattle/blood , Female , Tissue Distribution , Uterus/chemistry
2.
J Marital Fam Ther ; 32(4): 505-14, 2006 Oct.
Article in English | MEDLINE | ID: mdl-17120522

ABSTRACT

Juvenile Anorexia Nervosa (AN) is a severe problem both in terms of presenting symptomatology and its tendency toward chronicity. Researchers have consistently shown that family-based approaches are superior to individual approaches for the treatment of juvenile AN. This article addresses the capacity deficit of trained family therapists to treat this disease. The author reviews the effectiveness of Structural Family Therapy as a treatment of juvenile AN and the essential concepts and skills required by the family therapist to treat this disorder. The concepts of therapeutic crisis induction, enactment, and therapeutic intensity are discussed in detail. Recommendations are made for future research.


Subject(s)
Anorexia Nervosa/therapy , Family Therapy , Adolescent , Anorexia Nervosa/psychology , Communication , Conflict, Psychological , Curriculum , Education, Graduate , Family Therapy/education , Female , Humans , Male , Parent-Child Relations , Professional Competence , Psychotherapy , Treatment Outcome
4.
J Neurosci Res ; 62(4): 566-73, 2000 Nov 15.
Article in English | MEDLINE | ID: mdl-11070500

ABSTRACT

Ca(2+) and synaptotagmin (a Ca(2+)-binding protein that regulates axolemmal fusion of synaptic vesicles) play essential roles in the repair of axolemmal damage in invertebrate giant axons. We now report that neurites of a rat pheochromocytoma (PC12) cell line transected and maintained in a serum medium form a dye barrier (exclude an external hydrophilic fluorescent dye) and survive for 24-hr posttransection (based on morphology and retention of another hydrophilic dye internally loaded at 6-hr posttransection). Some (25%) transected neurites that form a dye barrier regrow. Most (83%) neurites transected in a saline solution containing divalent cations (PBS(++)) also exclude entry of an externally placed hydrophilic fluorescent dye at 15-min posttransection. In contrast, only 14 or 17% of neurites maintained in a divalent cation-free solution (PBS(=)) or in PBS(=) + Mg(2+), respectively, form a dye barrier. Neurites that do not form a dye barrier do not survive for 24 hr. When PC12 neurites are loaded with an antibody to squid synaptotagmin, most (81%) antibody-loaded neurites do not form a dye barrier, whereas most (>/=81%) neurites loaded with heat-inactivated antibody or preimmune IgG do form a barrier. These data show that: 1) transected neurites of PC12 cells have mechanism(s) for plasmalemmal repair (dye barrier formation and survival); 2) Ca(2+) is necessary for dye barrier formation, which occurs minutes after transection and is necessary for survival and regrowth; and 3) synaptotagmin is an essential mediator of barrier formation. The similarity in the requirements for plasmalemmal repair in this mammalian cell preparation with those reported previously for invertebrate axons suggests that mechanisms necessary for plasmalemmal repair have been conserved phylogenetically.


Subject(s)
Calcium-Binding Proteins , Calcium/metabolism , Cell Membrane/metabolism , Cell Survival/physiology , Membrane Glycoproteins/metabolism , Nerve Regeneration/physiology , Nerve Tissue Proteins/metabolism , Neurites/metabolism , PC12 Cells/metabolism , Animals , Axons/drug effects , Axons/metabolism , Axons/ultrastructure , Axotomy/adverse effects , Cell Membrane/drug effects , Cell Membrane/ultrastructure , Cell Survival/drug effects , Dextrans/pharmacology , Fluoresceins/pharmacology , Indicators and Reagents/pharmacology , Nerve Regeneration/drug effects , Neurites/drug effects , Neurites/ultrastructure , PC12 Cells/cytology , PC12 Cells/drug effects , Rats , Synaptotagmins
5.
Biophys J ; 79(4): 1883-90, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11023894

ABSTRACT

After axonal severance, a barrier forms at the cut ends to rapidly restrict bulk inflow and outflow. In severed crayfish axons we used the exclusion of hydrophilic, fluorescent dye molecules of different sizes (0.6-70 kDa) and the temporal decline of ionic injury current to levels in intact axons to determine the time course (0-120 min posttransection) of barrier formation and the posttransection time at which an axolemmal ionic seal had formed, as confirmed by the recovery of resting and action potentials. Confocal images showed that the posttransection time of dye exclusion was inversely related to dye molecular size. A barrier to the smallest dye molecule formed more rapidly (<60 min) than did the barrier to ionic entry (>60 min). These data show that axolemmal sealing lacks abrupt, large changes in barrier permeability that would be expected if a seal were to form suddenly, as previously assumed. Rather, these data suggest that a barrier forms gradually and slowly by restricting the movement of molecules of progressively smaller size amid injury-induced vesicles that accumulate, interact, and form junctional complexes with each other and the axolemma at the cut end. This process eventually culminates in an axolemmal ionic seal, and is not complete until ionic injury current returns to baseline levels measured in an undamaged axon.


Subject(s)
Axons/physiology , Animals , Astacoidea/physiology , Biophysical Phenomena , Biophysics , Fluorescent Dyes/pharmacokinetics , In Vitro Techniques , Ion Channels/metabolism , Microscopy, Confocal , Nerve Regeneration/physiology , Permeability
6.
J Neurobiol ; 44(4): 382-91, 2000 Sep 15.
Article in English | MEDLINE | ID: mdl-10945894

ABSTRACT

A damaged cell membrane is repaired by a seal that restricts entry or exit of molecules and ions to that of the level passing through an undamaged membrane. Seal formation requires elevation of intracellular Ca(2+) and, very likely, protein-mediated fusion of membranes. Ca(2+) also regulates the interaction between synaptotagmin (Syt) and syntaxin (Syx), which is thought to mediate fusion of synaptic vesicles with the axolemma, allowing transmitter release at synapses. To determine whether synaptic proteins have a role in sealing axolemmal damage, we injected squid and crayfish giant axons with an antibody that inhibits squid Syt from binding Ca(2+), or with another antibody that inhibits the Ca(2+)-dependent interaction of squid Syx with the Ca(2+)-binding domain of Syt. Axons injected with antibody to Syt did not seal, as assessed at axonal cut ends by the exclusion of extracellular hydrophilic fluorescent dye using confocal microscopy, and by the decay of extracellular injury current compared to levels measured in uninjured axons using a vibrating probe technique. In contrast, axons injected with either denatured antibody to Syt or preimmune IgG did seal. Similarly, axons injected with antibody to Syx did not seal, but did seal when injected with either denatured antibody to Syx or preimmune IgG. These results indicate an essential involvement of Syt and Syx in the repair (sealing) of severed axons. We suggest that vesicles, which accumulate and interact at the injury site, re-establish axolemmal continuity by Ca(2+)-induced fusions mediated by proteins such as those involved in neurotransmitter release.


Subject(s)
Axons/metabolism , Calcium-Binding Proteins , Membrane Fusion/physiology , Membrane Glycoproteins/metabolism , Membrane Proteins/metabolism , Nerve Tissue Proteins/metabolism , Synaptic Vesicles/metabolism , Animals , Antibodies/pharmacology , Antibody Specificity , Astacoidea , Axons/ultrastructure , Axotomy , Cell Membrane/metabolism , Decapodiformes , Fluorescent Dyes , Immunoblotting , In Vitro Techniques , Membrane Fusion/drug effects , Membrane Glycoproteins/antagonists & inhibitors , Membrane Proteins/antagonists & inhibitors , Nerve Tissue Proteins/antagonists & inhibitors , Neurons/drug effects , Neurons/physiology , Neurons/ultrastructure , Qa-SNARE Proteins , Synaptotagmins
7.
J Comp Neurol ; 416(2): 143-57, 2000 Jan 10.
Article in English | MEDLINE | ID: mdl-10581462

ABSTRACT

We describe structural changes at the cut ends of invertebrate myelinated earthworm giant axons beginning with the formation of a dye barrier (15 minutes posttransection or postcalcium addition) and ending with the formation of a neuritic outgrowth (2-10 days posttransection). The morphology of the cut end, and the location and morphological configuration of the dye barrier, were assessed by time-lapse confocal, fluorescence microscopy and by electron microscopy. During the interval from 15 to 35 minutes postcalcium addition, the dye barrier continuously migrated away from a cut axonal end; the dye barrier then remained stable for up to 5 hours. The size, packing density, and arrangement of membranous structures were correlated with changes in the dye barrier from 15 to 35 minutes postcalcium addition. During this interval, uptake of an externally placed hydrophilic dye by these membranous structures was also variable. After 35 minutes postcalcium addition, the membranous structures remained stable until they completely disappeared between 1 and 2 days posttransection. The disappearance of membranous structures always preceded neuritic outgrowth, which only arose from cut axonal ends. These results demonstrate that the dye barrier and associated membranous structures, which form after transection of earthworm giant axons, are very dynamic in the short term (35 minutes) with respect to their location and morphological configuration and suggest that axolemmal repair must be completed before neuritic outgrowth can occur.


Subject(s)
Axons/physiology , Giant Cells/physiology , Myelin Sheath/physiology , Neurites/physiology , Oligochaeta/ultrastructure , Animals , Axons/ultrastructure , Axotomy , Cell Membrane/physiology , Cell Membrane/ultrastructure , Coloring Agents , Giant Cells/ultrastructure , Myelin Sheath/ultrastructure , Neurites/ultrastructure , Time Factors
8.
Neurosci Lett ; 272(3): 147-50, 1999 Sep 17.
Article in English | MEDLINE | ID: mdl-10505602

ABSTRACT

After severance, axons can restore structural barriers that are necessary for recovery of their electrical function. In earthworm myelinated axons, such a barrier to dye entry is mediated by many vesicles and myelin-derived membranous structures. From time-lapse confocal fluorescence and DIC images, we now report that Ca2+ entry and not axonal injury per se initiates the processes that form a dye barrier, as well as the subsequent structural changes in this barrier and associated membranous structures. The time required to restore a dye barrier after transection also depends only on the time of Ca2+ entry.


Subject(s)
Axons/metabolism , Calcium/metabolism , Calcium/physiology , Coloring Agents/pharmacokinetics , Oligochaeta/metabolism , Animals , Axons/ultrastructure , Dextrans , Fluoresceins , Indicators and Reagents , Microscopy, Confocal , Microscopy, Fluorescence
9.
Community Ment Health J ; 35(3): 273-80, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10401896

ABSTRACT

Despite the dramatic growth of homelessness research, there have been no systematic assessments of consumer and provider preferences regarding the content of this research. Therefore, 87 clients and 28 staff of a homeless veterans program were administered a 15-item questionnaire requesting identification of the 5 "most" and 5 "least" important research topics. Staff and clients differed significantly on 6 items considered most important and 4 items considered least important. Clients wanted more research that focused on material needs, whereas staff preferences were more broadly distributed. The fact that appreciable data exist for many of the research topics that respondents identified as important raises concerns about the accessibility of homelessness research.


Subject(s)
Attitude , Community Participation , Ill-Housed Persons/psychology , Mental Health , Research , Veterans/psychology , Choice Behavior , Humans , Professional-Patient Relations , Surveys and Questionnaires
11.
Neurosci Lett ; 256(3): 123-6, 1998 Nov 13.
Article in English | MEDLINE | ID: mdl-9855355

ABSTRACT

After axonal injury, dye exclusion is often used as a measure of the re-establishment of a structural barrier. We now report that this use of dye exclusion is equivocal in two situations. (1) When a negatively-charged hydrophilic fluorescent dye (HFD) was placed in the physiological saline (PS) surrounding a crayfish medial giant axon (CMGA) before transection, this dye did not readily diffuse into the cut ends after transection whereas uncharged or neutralized dyes did do so. (2) When axoplasm flowed out of the cut ends of a transected squid giant axon (SGA), this outflow markedly slowed hydrophilic fluorescent dyes from diffusing into the cut ends. These anomalies suggest that dye exclusion by an injured axon does not always indicate that a structural barrier has formed. Therefore, dye assessments of axonal repair require control experiments that rule out anomalous exclusion due to dye interactions (biochemical and fluid dynamics) with components (axoplasm, axolemma, glial sheath, etc.) of the particular axon under study.


Subject(s)
Axonal Transport , Axons/physiology , Fluorescent Dyes/pharmacokinetics , Animals , Anions/pharmacokinetics , Astacoidea , Axonal Transport/drug effects , Axons/drug effects , Axotomy , Calcium/pharmacology , Decapodiformes , Nerve Regeneration/physiology , Time Factors
12.
Article in English | MEDLINE | ID: mdl-9646866

ABSTRACT

Identification of biomolecules in complex biological mixtures represents a major challenge in biomedical, environmental, and chemical research today. Chemical separations with traditional detection schemes such as absorption, fluorescence, refractive index, conductivity, and electrochemistry have been the standards for definitive identifications of many compounds. In many instances, however, the complexity of the biomixture exceeds the resolution capability of chemical separations. Biosensors based on molecular recognition can dramatically improve the selectivity of and provide biologically relevant information about the components. This review describes how coupling chemical separations with online biosensors solves challenging problems in sample analysis by identifying components that would not normally be detectable by either technique alone. This review also presents examples and principles of combining chemical separations with biosensor detection that uses living systems, whole cells, membrane receptors, enzymes, and immunosensors.


Subject(s)
Biosensing Techniques , Chemistry/methods , Pharmaceutical Preparations/isolation & purification , Animals , Biophysics/methods , Chemistry/instrumentation , Enzymes, Immobilized
13.
J Neurosci ; 18(11): 4029-41, 1998 Jun 01.
Article in English | MEDLINE | ID: mdl-9592084

ABSTRACT

Vesicles and/or other membranous structures that form after axolemmal damage have recently been shown to repair (seal) the axolemma of various nerve axons. To determine the origin of such membranous structures, (1) we internally dialyzed isolated intact squid giant axons (GAs) and showed that elevation of intracellular Ca2+ >100 microM produced membranous structures similar to those in axons transected in Ca2+-containing physiological saline; (2) we exposed GA axoplasm to Ca2+-containing salines and observed that membranous structures did not form after removing the axolemma and glial sheath but did form in severed GAs after >99% of their axoplasm was removed by internal perfusion; (3) we examined transected GAs and crayfish medial giant axons (MGAs) with time-lapse confocal fluorescence microscopy and showed that many injury-induced vesicles formed by endocytosis of the axolemma; (4) we examined the cut ends of GAs and MGAs with electron microscopy and showed that most membranous structures were single-walled at short (5-15 min) post-transection times, whereas more were double- and multi-walled and of probable glial origin after longer (30-150 min) post-transection times; and (5) we examined differential interference contrast and confocal images and showed that large and small lesions evoked similar injury responses in which barriers to dye diffusion formed amid an accumulation of vesicles and other membranous structures. These and other data suggest that Ca2+ inflow at large or small axolemmal lesions induces various membranous structures (including endocytotic vesicles) of glial or axonal origin to form, accumulate, and interact with each other, preformed vesicles, and/or the axolemma to repair the axolemmal damage.


Subject(s)
Axons/physiology , Calcium/pharmacology , Endocytosis/physiology , Synaptic Vesicles/physiology , Animals , Astacoidea , Axons/ultrastructure , Axotomy , Cell Communication/physiology , Cell Membrane/physiology , Decapodiformes , Endocytosis/drug effects , Intracellular Membranes/drug effects , Intracellular Membranes/physiology , Membrane Fusion/physiology , Microscopy, Electron , Microscopy, Fluorescence , Microscopy, Video , Neuroglia/physiology , Synaptic Vesicles/drug effects , Synaptic Vesicles/ultrastructure
14.
J Neurobiol ; 33(7): 945-60, 1997 Dec.
Article in English | MEDLINE | ID: mdl-9407015

ABSTRACT

Transected axons are often assumed to seal by collapse and fusion of the axolemmal leaflets at their cut ends. Using photomicroscopy and electronmicroscopy of fixed tissues and differential interference contrast and confocal fluorescence imaging of living tissues, we examined the proximal and distal cut ends of the pseudomyelinated medial giant axon of the earthworm, Lumbricus terrestris, at 5-60 min post-transection in physiological salines and Ca2+-free salines. In physiological salines, the axolemmal leaflets at the cut ends do not completely collapse, much less fuse, for at least 60 min post-transection. In fact, the axolemma is disrupted for 20-100 microm from the cut end at 5-60 min post-transection. However, a barrier to dye diffusion is observed when hydrophilic or styryl dyes are placed in the bath at 15-30 min post-transection. At 30-60 min post-transection, this barrier to dye diffusion near the cut end is formed amid an accumulation of some single-layered and many multilayered vesicles and other membranous material, much of which resembles delaminated pseudomyelin of the glial sheath. In Ca2+-free salines, this single and multilayered membranous material does not accumulate, and a dye diffusion barrier is not observed. These and other data are consistent with the hypothesis that plasmalemmal damage in eukaryotic cells is repaired by Ca2+-induced vesicles arising from invaginations or evaginations of membranes of various origin which form junctional contacts or fuse with each other and/or the plasmalemma.


Subject(s)
Axons/physiology , Myelin Sheath/physiology , Animals , Coloring Agents , Myelin Sheath/ultrastructure , Neuroglia/ultrastructure , Oligochaeta , Solubility , Styrene , Styrenes , Water/chemistry
15.
Proc Natl Acad Sci U S A ; 94(9): 4751-6, 1997 Apr 29.
Article in English | MEDLINE | ID: mdl-9114063

ABSTRACT

A barrier (seal) must form at the cut ends of a severed axon if a neuron is to survive and eventually regenerate. Following severance of crayfish medial giant axons in physiological saline, vesicles accumulate at the cut end and form a barrier (seal) to ion and dye diffusion. In contrast, squid giant axons do not seal, even though injury-induced vesicles form after axonal transection and accumulate at cut axonal ends. Neither axon seals in Ca2+-free salines. The addition of calpain to the bath saline induces the sealing of squid giant axons, whereas the addition of inhibitors of calpain activity inhibits the sealing of crayfish medial giant axons. These complementary effects involving calpain in two different axons suggest that endogenous calpain activity promotes plasmalemmal repair by vesicles or other membranes which form a plug or a continuous membrane barrier to seal cut axonal ends.


Subject(s)
Axons/physiology , Calpain/pharmacology , Cell Membrane/physiology , Membrane Fusion/drug effects , Animals , Astacoidea , Axons/drug effects , Cell Membrane/drug effects , Cysteine Proteinase Inhibitors/pharmacology , Decapodiformes , Electric Conductivity , Membrane Potentials , Species Specificity
16.
Proc Natl Acad Sci U S A ; 94(9): 4745-50, 1997 Apr 29.
Article in English | MEDLINE | ID: mdl-9114062

ABSTRACT

Crayfish medial giant axons (MGAs) transected in physiological saline form vesicles which interact with each other, pre-existing vesicles, and/or with the plasmalemma to form an electrical and a physical barrier that seals a cut axonal end within 60 min. The formation of this barrier (seal) was assessed by measuring the decay of injury current at the cut end; its location at the cut end was determined by the exclusion of fluorescent hydrophilic dye at the cut end. When a membrane-incorporating styryl dye was placed in the bath prior to axonal transection and a hydrophilic dye was placed in the bath just after axonal transection, many vesicles near the barrier at the cut axonal end had their limiting membrane labeled with the styryl dye and their contents labeled with the hydrophilic dye, indicating that these vesicles originated from the axolemma by endocytosis. This barrier does not form in Ca2+-free salines. Similar collections of vesicles have been observed at regions of plasmalemmal damage in many cell types. From these and other data, we propose that plasmalemmal lesions in most eukaryotic cells (including axons) are repaired by vesicles, at least some of which arise by endocytosis induced by Ca2+ inflow resulting from the plasmalemmal damage. We describe several models by which vesicles could interact with each other and/or with intact or damaged regions of the plasmalemma to repair small (1-30 microm) plasmalemmal holes or a complete transection of the plasmalemma.


Subject(s)
Axons/physiology , Cell Membrane/physiology , Animals , Astacoidea , Axons/ultrastructure , Calcium/metabolism , Cell Membrane/ultrastructure , Coloring Agents/metabolism , Endocytosis , Microscopy, Confocal , Microscopy, Interference , Models, Biological
17.
Science ; 272(5269): 1779-82, 1996 Jun 21.
Article in English | MEDLINE | ID: mdl-8650575

ABSTRACT

Gamma-aminobutyrate acid, L-glutamate, and N-methyl-D-aspartate were separated by capillary electrophoresis and detected by the use of whole-cell and outside-out patch-clamp techniques on freshly dissociated rat olfactory interneurons. These neuroactive compounds could be identified from their electrophoretic migration times, unitary channel conductances, and power spectra that yielded corner frequencies and mean single-channel conductances characteristic for each of the different agonist-receptor interactions. This technique has the sensitivity to observe the opening of a single ion channel for agonists separated by capillary electrophoresis.


Subject(s)
Glutamic Acid/analysis , Interneurons/chemistry , N-Methylaspartate/analysis , gamma-Aminobutyric Acid/analysis , Animals , Biosensing Techniques , Electrophoresis, Capillary , Glutamic Acid/isolation & purification , Ion Channels/physiology , N-Methylaspartate/isolation & purification , Olfactory Bulb/cytology , Patch-Clamp Techniques , Rats , Receptors, GABA/physiology , Receptors, Glutamate/physiology , Receptors, N-Methyl-D-Aspartate/physiology , Sensitivity and Specificity , gamma-Aminobutyric Acid/isolation & purification
18.
Anal Chem ; 68(7): 1181-6, 1996 Apr 01.
Article in English | MEDLINE | ID: mdl-8651490

ABSTRACT

A widespread limitation in using cell-based biosensors for repetitive chemical analysis is loss of agonist-induced response caused by receptor desensitization. We overcome this problem by scanning an array of immobilized cells underneath a capillary electrophoresis column outlet. In this way, electrophoretically fractionated components that exit the separation capillary are always directed onto cells previously unexposed to receptor agonists. To demonstrate this concept of response recovery using a scanning format, we have chosen the bradykinin B2 receptor system in the NG108-15 cell line, which is known to undergo desensitization. Whereas four subsequent injections of 250 microM bradykinin separated by 120 s are found to reduce the NG108-15 cell response markedly, scanning to new cells can fully restore the response during the separation. Furthermore, by pretesting individual NG108-15 cells for an agonist response and then later scanning back to the same cell, we achieved a 100% success rate in detecting bradykinin in subsequent electrophoretic separations.


Subject(s)
Biosensing Techniques , Glioma/ultrastructure , Neuroblastoma/ultrastructure , Receptors, Bradykinin/agonists , Animals , Bradykinin/pharmacology , Electrophoresis, Capillary/methods , Hybrid Cells , Mice , Rats , Receptor, Bradykinin B2
19.
Anal Chem ; 67(23): 4261-8, 1995 Dec 01.
Article in English | MEDLINE | ID: mdl-8633771

ABSTRACT

We report that 2,3-naphthalenedicarboxaldehyde reacts rapidly with glutathione and its precursor, gamma-glutamylcysteine, to form highly fluorescent derivatives under physiological conditions. In contrast to previous accounts of 2,3-naphthalenedicarboxaldehyde labeling of primary amines, no additional CN- ion or any other additional nucleophile is required. The fluorescence spectral properties of the chromophores (lambda exc max = 472 nm, lambda em max = 528 nm) make these derivatives amenable to excitation and detection by optical instrumentation that is optimized for fluorescein wavelengths. This selective labeling chemistry enabled quantitative determination and histochemical localization of glutathione in neurobiological samples. Intracellular glutathione was labeled by incubating cultured cells or cell suspensions in a 2,3-naphthalenedicarboxaldehyde-supplemented, DMSO-containing physiological buffer (pH = 7.4) for 2-10 min. Applications include imaging of cultured NG 108-15 cells (mouse neuroblastoma x rat glioma) and primary glial and neuronal cell cocultures (rat hippocampus) using epiluminescent and confocal fluorescence microscopy. Quantitative determination of glutathione in single NG 108-15 cells was accomplished using laser-induced fluorescence detection and capillary electrophoresis.


Subject(s)
Glutathione/analysis , Animals , Cells, Cultured , Electrophoresis, Capillary , Mice , Microscopy, Confocal , Microscopy, Fluorescence , Naphthalenes , Rats
20.
Biophys J ; 69(6): 2458-66, 1995 Dec.
Article in English | MEDLINE | ID: mdl-8599652

ABSTRACT

A steady, spontaneous current oscillation (1 nA p-p) occurs in voltage-clamped, isolated ampullary organs (canal, ampulla, and nerve) from skates (Raja). Spectral analysis showed that energy in the oscillation was confined to a narrow band of frequencies (3 Hz) about a fundamental frequency (32 Hz at 20 degrees C) and in harmonics. The frequency of the oscillation was temperature dependent (increasing from 21 to 33 Hz for increases in temperature from 13 degrees C to 21 degrees C). The addition of 0.5 microM tetrodotoxin to the basal side of the ampullary epithelium eliminated afferent nerve activity but had no effect on the epithelial oscillation, indicating that the oscillation is not generated or induced by afferent nerve activity. Nitrendipine (2 microM) added to the solution bathing the basal side of the ampullary epithelium abolished the oscillation rapidly (within minutes), but a steady-state negative conductance (i.e., real part of the complex admittance < 0) generated by the preparation remained for 36 min. Conversely, nitrendipine (50 microM) added to the perfusate (artificial sea water) of the apical side eliminated the negative conductance rapidly (18.5 min) but had no effect on the spontaneous oscillation for more than 1 h. The effect and the elapsed time for an effect of nitrendipine after separate applications to the basal and apical membrane surfaces of ampullary epithelium suggest that 1) the negative conductance and the oscillation are generated independently in apical and basal membranes, respectively, and 2) both processes involve L-type Ca channels. Furthermore, the addition of tetraethylammonium (2 mM) to the basal side eliminated both the oscillation and the postsynaptic response to voltage clamps (< or = 100 microV) of the ampullary epithelium in the operational voltage range of the afferent nerve. This result suggests that the basal membrane oscillation functions in neurotransmitter release from presynaptic (basal) membranes.


Subject(s)
Sensory Receptor Cells/physiology , Afferent Pathways/physiology , Animals , Cell Membrane/drug effects , Cell Membrane/physiology , Electric Conductivity , Epithelium/drug effects , Epithelium/physiology , In Vitro Techniques , Membrane Potentials , Nitrendipine/pharmacology , Oscillometry , Patch-Clamp Techniques , Sensory Receptor Cells/drug effects , Skates, Fish , Tetrodotoxin/pharmacology
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