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51.
52.
Lawrence W. Fitzgerald‡ Lynne Kaplinsky† Harold K. Kimelberg†‡ 《Journal of neurochemistry》1990,55(6):2008-2014
The oxidative deamination of serotonin (5-HT) to 5-hydroxyindoleacetic acid (5-HIAA) by rat primary astrocyte cultures was investigated in intact cells using HPLC. All detectable 5-HIAA accumulated in the extracellular medium, and its rate of production was proportional to the 5-HT concentration over the tested range of 5 x 10(-7) to 10(-4) M. At 5 x 10(-7) M 5-HT, intracellular 5-HT was detectable only in astrocytes treated with monoamine oxidase (MAO) inhibitors. These findings are consistent with the idea that 5-HT taken up into astrocytes is not stored for re-release, but is rapidly metabolized to 5-HIAA, which is then extruded from the cell. At 5 x 10(-7) M 5-HT, 5-HIAA formation in intact cells was blocked 63% by the selective high-affinity 5-HT uptake inhibitor fluoxetine. 5-HT oxidation to 5-HIAA is carried out principally by MAO-A, because clorgyline was more effective at inhibiting the production of 5-HIAA than was pargyline. Radioenzymatic determinations of MAO activity in cell homogenates supported these findings, because under these conditions clorgyline was 1,000-fold more effective than pargyline at inhibiting MAO activity toward 14C-labelled 5-HT. However, the relatively selective MAO-B substrate beta-phenylethylamine (PEA) was also oxidized, showing that these cultures also contained MAO-B activity; the Km values for MAO-A oxidation of 5-HT and MAO-B oxidation of PEA were 135 and 45 microM, and Vmax values were 88 and 91 nmol/mg of total cell protein/h, respectively. Higher concentrations of PEA (greater than 20 microM) were oxidized by both MAO-A and MAO-B isozymes.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
53.
Vanessa R. Panz Paul Ruff Barry I. Joffe Mary-Anne Kedda Harold C. Seftel 《Human genetics》1996,97(4):438-440
The frequency of DNA polymorphisms in the tyrosine kinase domain (exons 17–21) of the insulin receptor gene was assessed
in 30 black and 30 white South Africans, using single-stranded conformation polymorphism and direct sequencing analysis. A
comparison of the frequencies of the normal versus the combined polymorphic alleles, found only in exon 17, showed a significant
difference between black and white groups (P = 0.037).
Received: 25 May 1995 / Revised: 1 September 1995 相似文献
54.
Potassium and Taurine Release Are Highly Correlated with Regulatory Volume Decrease in Neonatal Primary Rat Astrocyte Cultures 总被引:2,自引:0,他引:2
Domenico Vitarella †Darryl J. DiRisio †Harold K. Kimelberg Michael Aschner 《Journal of neurochemistry》1994,63(3):1143-1149
Abstract: Neonatal rat primary astrocyte cultures were swollen by exposure to hypotonic buffer. Using an electrical impedance method for determination of cell volume coupled with on-line measurements of efflux of radioactive ions or amino acids, we have investigated the role of K+ (using 86 Rb), taurine, and d -aspartate (an analogue of glutamate) in regulatory volume decrease (RVD). Addition of 1 m M quinine, 10 µ M nimodipine, 100 µ M BAPTA-AM, 10 µ M trifluoperazine, or a calcium-free buffer significantly ( p < 0.0001) inhibited RVD. This was accompanied by inhibition of 86 Rb release but an increase in d -[3 H]-aspartate release, which was proportional to the degree to which RVD was inhibited. These results support a regulatory role for calcium in RVD and show that inhibition of calcium entry from the extracellular fluid, intracellular calcium sequestration, inhibition of calcium-activated K+ channels, and inhibition of calmodulin all inhibit RVD. Because d -[3 H]aspartate efflux profiles increase as RVD is inhibited, it is unlikely that d -aspartate release is a main determinant of RVD. In contrast, [3 H]taurine release was increased by 1 m M quinine and inhibited by 10 µ M trifluoperazine. The net release of K+ and taurine is highly correlated with the degree of RVD, implicating a regulatory role for both K+ and taurine release in RVD. 相似文献
55.
From the cambial stage onwards, the symplasmic autonomy of sieve element/companion cell complexes (SE/CC-complexes) was followed in stems of Lupinus luteus L. by microinjection techniques. The membrane potential and the symplasmic autonomy of the mature SE/CC-complex was measured in successive internodes. A microelectrode was inserted into SE/CC-complexes or phloem parenchyma cells (PPs) and, after stabilization of the membrane potential, the membrane-impermeant fluorescent dye Lucifer Yellow CH (LYCH) was injected intracellullary. The plasmodesmata of the cambial SE/ CC precursor were gradually shut off at all interfaces beginning at the walls to be transformed into sieve plates. In the course of maturation, symplasmic discontinuity was maintained at the longitudinal walls of the complex. In the transverse walls of the SE, wide sieve pores were formed giving rise to longitudinal multicellular symplasmic domains of SE/CC-complexes. Symplasmic isolation of the files of mature SE/CC-complexes was demonstrated in several ways: (i) the membrane potential of the SE/CC-complexes (between -100 mV and -130 mV) was consistently more negative than that of the PPs (between-50 and -100 mV), (ii) No exchange of LYCH was observed between SE/CC-complexes and the PPs. Lucifer Yellow CH injected into the SEs exclusively moved to the associated CCs and to other SE/CC-complexes whereas LYCH injected into the PPs was only displaced to other PPs. (iii) The electrical coupling ratio between adjacent PPs was ten times higher than that between SE/CC-complex and PP. A gradient in the membrane potential of the SE/CC-complexes along the stem was not conclusively demonstrated.Abbreviations LYCH
Lucifer Yellow CH
-
membrane potential
- PMF
proton-motive force
- PP
phloem parenchyma cell
- SE/CC-complex
sieve element/companion cell complex
- SR-G
sulphorhodamine G 相似文献
56.
Summary Human embryonic kidney cells (293) were transfected with a construct containing human factor X cDNA and selected for G418 resistance. The level of expression of recombinant factor X in serum-free medium was 4 to 5 g/ml. Purified recombinant factor X had a molecular size identical to that of normal plasma factor X. Amino-terminal sequencing revealed normal processing cleavages. The -carboxy Glu and -OH Asp content of the recombinant factor X was close to 90% of the expected levels of these post-translational residues. The specific activity of recombinant factor X was about 95% of that of plasma factor X in three plasma-based clotting assays. This report demonstrates that 293 cells can produce a high level of biologically active factor X and describes a visual criterion for verifying the transfection process.Abbreviations FX
factor X
- rFX
recombinant factor X
- DMEM
Dulbecco's modified Eagle's medium
- RVV-X
Russell's viper venom
- SDS-PAGE
sodium dodecyl sulfatepolyacrylamide gel electrophoresis
- Gla
-carboxy glutamic acid 相似文献
57.
Souvik Sen Harold Goldman Marilyn Morehead Sharon Murphy John W. Phillis 《Free radical biology & medicine》1994,16(6)
Traumatic brain injury (TBI) is one of the important causes of mortality and morbidity. The pathogenesis of the underlying brain dysfunction is poorly understood. Recent data have suggested that oxygen free radicals play a key role in the primary and secondary processes of acute TBI. We report direct electron spin resonance (ESR) evidence of hydroxyl (·OH) radical generation in closed-head injury of rats. Moderate brain concussion was produced by controlled and reproducible mechanical, fixed, closed-head injury. A cortical cup was placed over one cerebral hemisphere within 20 min of the concussion, perfused with artificial cerebrospinal fluid (aCSF) containing the spin trap agent pyridyl-N-oxide-tert-butyl nitrone (POBN, 100 mM), and superfusate samples collected at 10 min intervals for a duration up to 130 min post brain trauma. In addition, POBN was administered systematically (50 mg/kg body wt.) 10 min pretrauma and 20 min posttrauma to improve our ability to detect free radicals. ESR analysis of the superfusate samples revealed six line spectra (αN = 15.4 and αβH = 2.5 G) characteristic of POBN-OH radical adducts, the intensity of which peaked 40 min posttrauma. The signal was undetectable after 120 min. Administration of α-phenyl-tert-butyl-nitrone (PBN), a spin adduct forming agent systemically (100 mg/kg body wt. IP 10 min prior to concussion) alone or along with topical PBN (100 mM PBN in aCSF),6significantly (P< 0.001) attenuated the ESR signal, suggesting its possible role in the treatment of TBI. 相似文献
58.
Elena P. Sawin Harold B. Dowse Melanie J. Hamblen-Coyle Jeffery C. Hall Marla B. Sokolowski 《Journal of Insect Behavior》1994,7(3):249-262
We examined the locomotor activity ofDrosophila melanogaster for the existence of circadian rhythms, using the wild type and two mutants of theperiod (per) gene,per
o andper
s. This was accomplished using a newly described apparatus for the recording and measurement of larval path lengths over a 96-h test period. None of the larvae examined exhibited appreciable diel rhythms under cycling conditions of light or temperature. Larvae were also not rhythmic under free-running conditions. Our results suggest that theper gene does not influence an observable locomotor behavioral phenotype in the larval stage of development. 相似文献
59.
The Delaware Bay is characterized as having greater nutrient and turbidity levels than the Chesapeake Bay. In reference to these differences, a one year study was conducted to identify any similarities and differences in the phytoplankton populations in these estuaries. The results indicated patterns of similarity in the diatom composition, with the total phytoplankton assemblage forming two site groups along a salinity gradient in each bay. These site groups were associated with stations located in the tidal fresh-oligohaline and meso-polyhaline regions of both estuaries. The seasonal concentrations of diatoms and total phytoplankton in both of these regions were higher in the Chesapeake Bay.Subtle differences between the two estuaries include a more diversified and abundant assemblage of neritic phytoplankters (including dinoflagellates) are present in the lower Chesapeake Bay. In contrast, a diatom dominated community is more characteristic of Delaware Bay. It is suggested the entry of neritic species into lower regions of the estuaries was enhanced by the reduced amount of rainfall and flow rates that occurred during the study period. The greater success of neritic species in the Chesapeake Bay is attributed to the lower turbidity of that estuary compared to Delaware Bay. 相似文献
60.
Sharon X. Chen Charles C. Hardin Harold E. Swaisgood 《Journal of Protein Chemistry》1993,12(5):613-625
Incubation of -lactoglobulin with immobilized trypsin at 5–10°C results in a time-dependent release of several fragments of the core domain in yields approaching 15%. Digests were fractionated by ion-exchange chromatography with a Mono Q HR5/5 column and analyzed after disulfide reduction by polyacrylamide gel electrophoresis in sodium dodecylsulfate. Three fragments with approximate molecular weights of 13.8, 9.6, and 6.7 kD were identified. The fraction from ion-exchange chromatography yielding the 6.7 kD fraction after disulfide reduction was further characterized because it was most homogeneous and gave the highest yield. The C-terminal cleavage site of the 6.7 kD core fragment appeared to be Lys100 or Lys101 as determined by C-terminal amino acid analysis. The exact masses, after reduction with dithiothreitol, are 6195 and 6926 as determined by laser desorption mass spectrometry, corresponding to residues 48–101 and 41–100. Prior to reduction, -lactoglobulin C-terminal residues 149–162 are connected to these core domain fragments as shown by C-terminal analysis and mass spectrometry. Structural studies indicate that these 7.9 and 8.6 kD core domain fragments released by immobilized trypsin retain much of their native structure. CD spectra indicate the presence of antiparallel -sheet structure similar to the native protein but the -helix is lost. Spectra in the aromatic region indicate the existence of tertiary structure. Moreover, structural transitions in urea are completely reversible as measured by CD spectra, although the extrapolated G
D
H20
and the urea concentration at the transition midpoint are lower than for the native protein. The core domain fragments also display apH-dependent binding to immobilizedtrans-retinal as does intact protein. A single endotherm is obtained for both core domain fragments and native protein upon differential scanning calorimetry, but again, the domain is less stable as indicated by a transition peak maxima of 56.9°C as compared with 81.1°C for native protein.Abbreviations used: CD, circular dichroism; CPG, controlled pore glass; DSC, differential scanning calorimetry; DTT, dithiothreitol; FPLC, fast flow liquid chromatography; HPLC, high-performance liquid chromatography; PITC, phenylisothiocyanate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TEA, triethylamine; UV, ultraviolet. 相似文献