Dealing with the public: issues for the intern.
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Communication↗
Biomedical subjects
G Matthews
Publications and source records attributed to G Matthews.
Dynamics of synaptic vesicle fusion and membrane retrieval in synaptic terminals.
Communication among neurons occurs at specialized synaptic junctions, where neurotransmitter is released via calcium-dependent exocytosis from the synaptic terminal of the presynaptic cell onto the postsynaptic target neuron. Here we exploit the unique properties of giant synaptic terminals of bipolar neurons from goldfish retina to establish the kinetics and calcium-dependence of exocytosis, and the characteristics of membrane retrieval following secretion in presynaptic terminals. Simultaneous patch-clamp, calcium-indicator dye and time-resolved capacitance measurements reveal that activation of calcium current drives secretion at a rapid rate of about 10,000 vesicles per s and the calcium level necessary to drive secretion is locally greater than 50 microM. Two components of membrane retrieval were observed following secretory stimulation. After strong stimulation, capacitance returned to rest with a time constant of about 30 s, but after weaker stimuli recovery was much faster, with a time constant of about 2 s. Secretion in a vertebrate central nervous system neuron was thus found to differ substantially from that in other secretory cells in its rapid rate of vesicle fusion, requirement for high levels of intracellular calcium, and the high speed and completeness of membrane retrieval. These distinctive features reflect the specialization of neuronal synaptic terminals for rapid and focally directed release of neurotransmitter.
Animals↗
Dopamine enhances Ca2+ responses in synaptic terminals of retinal bipolar neurons.
The effect of dopamine on depolarization-induced Ca2+ influx was studied using the fluorescent Ca2+ indicator fura-2 in synaptic terminals of bipolar neurons from gold-fish retina. Dopamine reversibly enhanced the rise in intracellular Ca2+ elicited by elevated external potassium. The enhancement was slowly reversible. The effect of dopamine was mimicked by forskolin and CPT-cAMP, a membrane-permeant analog of cAMP. However, 1,9-dideoxyforskolin, a forskolin analog that does not activate adenylyl cyclase, was ineffective. This suggests that dopamine, via cAMP, regulates the rise in presynaptic Ca2+ concentration in response to depolarization, potentially enhancing transmitter release.
Animals↗
Autocatalytic maturation of the prohormone convertase PC2.
PC2 is a member of the eukaryotic family of subtilisin-like proteases, which is thought to participate in the processing of prohormones and proneuropeptides in neuroendocrine cells. PC2 is synthesized as a 69-kDa prepropolypeptide. The NH2-terminal signal sequence is removed during segregation within the endoplasmic reticulum, where glycosylation occurs to generate a 75-kDa propolypeptide. A combination of site-directed mutagenesis and a cell-free translation/translocation system from Xenopus eggs was used to investigate the processing of the pro-PC2 precursor. The 75-kDa polypeptide underwent slow cleavage after the sequence Arg-Lys-Lys-Arg84 to generate a 68-kDa mature enzyme. Cleavage was blocked when the tetrabasic sequence was deleted (PC2M3) or when the active site Asp142 was changed to Asn (PC2M4). This latter observation suggested that cleavage of the 75-kDa propolypeptide to the mature 68-kDa enzyme was autocatalytic. Incubation of the PC2M4 mutant with the wild type PC2 precursor resulted in cleavage of both the wild type polypeptide and the catalytically inactive PC2M4 mutant. This indicates that cleavage could occur through an intermolecular reaction. The results also demonstrate that the novel Xenopus egg extract translation/translocation system represents a powerful cell-free method for studying proteolytic processing of propolypeptides.
Amino Acid Sequence↗
The expression of the 'pre-menstrual syndrome' in measures of mood and sustained attention.
Twenty-eight women performed two demanding sustained attention tasks in the premenstrual and intermenstrual phases of the menstrual cycle. Mood was assessed before and after performance. No effects of phase were found on perceptual sensitivity and response criterion, but response time on the less demanding of the two tasks was slower in the premenstrual phase. There were no effects of phase on pre-task mood, but following performance, premenstrual women were lower in energy and in pleasantness of mood. Moods varied with phase irrespective of usage of the contraceptive pill, but effects on response time were stronger in non-pill users. Performance data suggest that speed of visual discrimination varies with the cycle, but this finding is of little practical significance. The mood data suggest that the affective correlates of the cycle vary with situational stress, and, in the workplace, it may be desirable to reduce stress and workload during the premenstrual phase.
Adult↗
Presynaptic inhibition by GABA is mediated via two distinct GABA receptors with novel pharmacology.
Mechanisms of presynaptic inhibition were examined in giant presynaptic terminals of retinal bipolar neurons, which receive GABAergic feedback synapses from amacrine cells. Two distinct inhibitory actions of GABA are present in the terminals: a GABAA-like Cl conductance and a GABAB-like inhibition of voltage-dependent Ca current. Both of the receptors underlying these actions have unusual pharmacology that fits neither GABAA nor GABAB classifications. The GABA-activated Cl conductance was not blocked by the classical GABAA antagonist bicuculline, while the inhibition of Ca current was neither mimicked by the GABAB agonist baclofen nor blocked by the GABAB antagonist 2-hydroxysaclofen. The "GABAC" agonist cis-4-aminocrotonic acid (CACA) both activated the Cl conductance and inhibited Ca current, but the inhibition of Ca current was observed at much lower concentrations of CACA (< 1 microM) than was the activation of the Cl conductance (K1/2 = 50 microM). Thus, by the criterion of being insensitive to both bicuculline and baclofen, both GABA receptors qualify as potential GABAC receptors. However, it is argued on functional grounds that the two GABA receptors coupled to Cl channels and to Ca channels are best regarded as members of the GABAA and GABAB families, respectively.
Animals↗
Ca2+ and Mn2+ influx through receptor-mediated activation of nonspecific cation channels in mast cells.
Whole-cell patch-clamp recordings of membrane currents and Fura-2 measurements of free intracellular calcium concentration ([Ca2+]i) were used to study calcium influx through receptor-activated cation channels in rat peritoneal mast cells. Cation channels were activated by the secretagogue compound 48/80, whereas a possible concomitant Ca2+ entry through pathways activated by depletion of calcium stores was blocked by dialyzing cells with heparin. Heparin effectively suppressed the transient Ca2+ release induced by 48/80 and abrogated inositol 1,4,5-trisphosphate-induced calcium influx without affecting activation of 50-pS cation channels. There was a clear correlation between changes in [Ca2+]i and the activity of 50-pS channels. The changes in [Ca2+]i increased with elevation of extracellular Ca2+. At the same time, inward currents through 50-pS channels were diminished as more Ca2+ permeated. This effect was due to a decrease in slope conductance and a reduction in the open probability of the cation channels. In physiological solutions, 3.6% of the total current was carried by Ca2+. The cation channels were not only permeable to Ca2+ but also to Mn2+, as evidenced by the quench of Fura-2 fluorescence. Mn2+ current through 50-pS channels could not be resolved at the single-channel level. Our results suggest that 50-pS cation channels partially contribute to sustained increases of [Ca2+]i in mast cells following receptor activation.
Animals↗
Secretion and mesoderm-inducing activity of the TGF-beta-related domain of Xenopus Vg1.
Vg1 is a maternal mRNA localized to the vegetal hemisphere of Xenopus embryos during blastula stages, a region responsible for the induction of mesoderm in the adjacent marginal zone. Its homology to the transforming growth factor-beta family, which includes several proteins with mesoderm-inducing activity, suggests a role for Vg1 as an endogenous mesoderm-inducing factor. However, expression of Vg1 protein in the animal hemisphere, following injection of synthetic mRNA, has no effect on development, and isolated animal caps are not mesodermalized. It is shown that Vg1 protein fails to form dimers and is not processed to release the putative bioactive domain. Furthermore it is shown that the N-terminal signal peptide of Vg1 is not cleaved following translocation into the ER, which may explain the failure of this protein to dimerize. To explore the role of Vg1 in amphibian development, a fusion protein has been made of the preproregion of Xenopus bone morphogenetic protein-4 and the putative bioactive C-terminal domain of Vg1. This fusion protein forms dimers and the C-terminal domain of Vg1 is secreted. Injection of this construct into Xenopus embryos induces the formation of a second dorsal axis and isolated animal caps are mesodermalized. The results are consistent with a role for Vg1 in mesoderm induction during Xenopus development.
Amino Acid Sequence↗
Chloride current activated by swelling in retinal pigment epithelium cells.
A membrane conductance activated by cell swelling was characterized in cells of the retinal pigment epithelium (RPE). Manipulations of internal and external Cl concentration revealed that the conductance is permeable to Cl and somewhat permeable to the gluconate anion used for Cl substitution (ratio of gluconate to Cl permeability approximately 0.1). The conductance was blocked by the Cl channel blockers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid in a manner consistent with open-channel blockade. Both the onset and recovery of the Cl current following a transient increase in cell volume were slow. This suggests that activation of the current depends on some internal signal rather than directly on membrane stretch. Experiments to examine a possible role for intracellular Ca concentration ([Ca]i) in regulation of the current demonstrated that an increase in [Ca]i was not involved in the linkage between swelling and Cl current; activation of the current was unaffected by the calcium-buffering conditions, the current could not be activated by large increases in [Ca]i elicited by ionomycin, and no changes in [Ca]i were observed to be associated with swelling. RPE cells normally experience changes in the volume and ionic composition of the extracellular subretinal space during changes in illumination conditions; therefore, the volume-sensitive Cl conductance may play a role in volume regulation in the RPE in response to these extracellular changes and/or in transepithelial fluid transport.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
Cognitive predictors of vigilance.
Three studies are reported of individual differences in performance of high event rate vigilance tasks. In each study, measures of attentional resource availability and elementary cognitive components of sustained attention were correlated with vigilance performance. It was predicted that correlates of vigilance should vary with two parameters of the task: the type of target discrimination required (simultaneous or successive) and the type of stimuli used (sensory or symbolic). Speed and accuracy of controlled, resource-demanding visual search predicted overall perceptual sensitivity on three out of four successive tasks used, but only one out of four simultaneous tasks, partially confirming the hypothesis that successive tasks are more strongly resource-limited than simultaneous tasks. Other correlates of overall level vigilance did not appear to vary systematically with task parameters. Few correlates of temporal decrement in perceptual sensitivity were found, possibly for statistical reasons. The data suggest that measures of controlled search may be useful in predicting vigilance on certain real-world tasks, although a substantial part of the variance of vigilance tasks may not be predictable from short cognitive tasks.
Adult↗
In vitro permeability of furcation dentin in permanent teeth.
Extracted unerupted permanent third molars with the occlusal half of the crown and apical half of the roots removed were cemented to Plexiglas blocks. Using a positive pressure system in which the movement of fluid across the dental tubules could be measured, the permeability of furcation dentin was measured before and after alteration of the furcation thickness. Subsequently, measurements of reduction in thickness of cementum and dentin were performed. Furcation dentin permeability was found to increase as the cementum and dentin thickness was reduced and the smear layer removed. The permeability values obtained for the furcation dentin were similar to those found in radicular dentin in general, indicating that root dentin has a low permeability and that it has good barrier properties. The data would suggest that any bone resorption seen under the furcation region of permanent molars is more likely to be due to the presence of accessory canals than due to permeation directly through furcation hard tissues.
Adolescent↗
Blockers of potassium channels reduce the outward dark current in rod photoreceptor inner segments.
The dark current of single isolated toad rods was monitored by drawing either the inner segment or the outer segment into a suction electrode. The potassium-channel blockers tetraethylammonium (TEA) and 3,4-diaminopyridine (DAP) reduced the amplitude of the dark current when applied to the inner segment. Both drugs were less effective when applied to the outer segment, suggesting that they act at the inner segment to block part of the outward path for the dark current. In addition, DAP affected the kinetics of the light response, possibly by affecting internal pH.
4-Aminopyridine↗
Substance P modulates calcium current in retinal bipolar neurons.
Retinal bipolar cells are non-spiking interneurons that relay information from photoreceptors to amacrine and ganglion cells. In turn, bipolar cells receive extensive synaptic feedback from amacrine cells, some of which contain neuropeptides, including substance P. We have examined the effect of substance P on single bipolar neurons isolated from goldfish retina and find that substance P (0.1-1 nM) produced a voltage-dependent inhibition of calcium current in these cells. The inhibition was strongest at negative potentials, with the peak suppression occurring at -20 to -30 mV; at potentials positive to 0 mV, there was little effect on calcium current. Thus, the net effect was to shift the voltage range of activation of calcium current toward more positive potentials. The inhibition of calcium current by substance P required GTP in the patch pipette and was blocked by internal GDP-beta-S. Similar effects on calcium current were observed with somatostatin and metenkephalin, which are also found in amacrine cells.
Animals↗
Calcium influx and calcium current in single synaptic terminals of goldfish retinal bipolar neurons.
1. The calcium influx pathway in large synaptic terminals of acutely isolated bipolar neurons from goldfish retina was characterized using Fura-2 measurements of intracellular calcium and patch-clamp recordings of whole-cell calcium current. 2. Depolarization of bipolar cells with high [K+]o resulted in a sustained, reversible increase in [Ca2+]i in both synaptic terminals and somata. Removal of external calcium abolished the response, as did the addition of 200 microM-cadmium to the bathing solution, indicating that the rise in [Ca2+]i was due to entry of external calcium. Dihydropyridine blockers of voltage-gated Ca2+ channels also blocked the influx, and the Ca2+ channel agonist Bay K 8644 potentiated influx, implicating voltage-activated, dihydropyridine-sensitive channels in the influx pathway. 3. Under voltage clamp, depolarization from a holding potential of -60 mV evoked a slowly inactivating inward current that began to activate at -50 to -40 mV and reached a maximal amplitude between -20 and -15 mV. This current was identified as a calcium current because it decreased when the extracellular calcium concentration was lowered, increased when barium was the charge carrier, and was blocked by 200 microM-external cadmium. The current was substantially blocked by 1 microM-nitrendipine and potentiated by 0.1 microM-Bay K 8644, as expected for L-type Ca2+ channels; it was unaffected by omega-conotoxin. No evidence for transient or rapidly inactivating Ca2+ current was found. 4. At a given level of potassium depolarization, both the amplitude and the speed of increase in [Ca2+]i were greater in synaptic terminals than in somata. For instance, depolarization by 32.6 mM-potassium caused an increase in intracellular calcium of 400 +/- 23 nM in terminals and 180 +/- 20 nM in somata (mean +/- S.E.M., n = 73 terminals, n = 30 somata), with maximal rates of change of 40 +/- 3 and 12 +/- 2 nM/s, respectively. 5. The contribution of terminal and somatic currents to the total whole-cell Ca2+ current was determined under voltage clamp by local application of calcium or of blocking agents. While there was no qualitative difference between currents in terminals and somata, synaptic terminals accounted for 64 +/- 3% (mean +/- S.E.M., n = 12) of the total whole-cell calcium current, and somata accounted for 39 +/- 2%. Thus, the density of Ca2+ current was higher in the terminal, accounting for the greater magnitude and speed of Ca2+ influx observed in terminals in Fura-2 experiments.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Secretory and inductive properties of Drosophila wingless protein in Xenopus oocytes and embryos.
Like its vertebrate homologues, Xenopus wnt-8 and murine wnt-1, we find that Drosophila wingless (wg) protein causes axis duplication when overexpressed in embryos of Xenopus laevis after mRNA injection. In many cases, the secondary axes contain eyes and cement glands, which reflect the induction of the most dorsoanterior mesodermal type, prechordal mesoderm. We show that the extent of axis duplication is dependent on the embryonic site of expression, with ventral expression leading to a more posterior point of axis bifurcation. The observed duplications are due to de novo generation of new axes as shown by rescue of UV-irradiated embryos. The true dorsal mesoderm-inducing properties of wg protein are indicated by its ability to generate extensive duplications after mRNA injection into D-tier cells of 32-cell embryos. As revealed by lineage mapping, the majority of these D cell progeny populate the endoderm; injections into animal blastomeres at this stage are far less effective in inducing secondary axes. However, when expressed in isolated animal cap explants, wg protein induces only ventral mesoderm, unless basic fibroblast growth factor is added, whereupon induction of muscle and occasionally notochord is seen. We conclude that in intact embryos, wg acts in concert with other factors to cause axis duplication. Immunolocalisation studies in embryos indicate that wg protein remains localised to the blastomeres synthesizing it and has a patchy, often perinuclear distribution within these cells, although some gets to the surface. In oocytes, the pool of wg protein is entirely intracellular and relatively unstable. When the polyanion suramin is added, most of the intracellular material is recovered in the external medium.
Animals↗
Maximizing productivity of new associate physicians: proven strategies.
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Humans↗
Getting paid for your hospital work.
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Humans↗
Digesting government's regulatory alphabet soup.
The impact of the governmental regulatory acronyms implemented during this year will prove to be very similar to how implementation of the Prospective Payment System (PPS) via DRGs affected hospitals a decade ago. Those hospitals that realized the dimensional shift in their industry and made prudent business decisions and adjustments have not only survived their era of payment reform but have also flourished. Those hospitals which did not recognize the regulatory changes, or chose to ignore the changes, have not succeeded, many paying the ultimate price of business failure. The impact on physicians will be quite similar. Physicians that adopt proven, sound, business principles and accept the absolute need to be informed and pro-active, will not only succeed, but they too, will flourish. Physicians who choose otherwise, quite frankly, risk practice failure. A few years ago, the American Hospital Association published what I believe is the ultimate statement of health care regulatory bureaucracy. The publication boasted 80 pages, containing more than 2,000 entries. Its title was Acronyms and Initialisms in Health Care Administration. With the addition of the regulatory acronyms implemented during 1992, the size of that publication has probably more than doubled by now. Being able to digest this regulatory alphabet soup has its benefits; not only will physicians feel more comfortable in their knowledge of being in regulatory compliance, it may also help physicians focus on the best methods of managing their practices and ultimately, their careers.
Efficiency↗