Biomedical subjects

L Li

Publications and source records attributed to L Li.

At least 1,405 records · Page 78Linked to original sources

PubMed1996

Effect of C-peptide administration on whole body glucose utilization in STZ-induced diabetic rats.

Recent studies suggest that C-peptide stimulates glucose transport in isolated skeletal muscle. In order to determine the effect of C-peptide on whole body glucose utilization, streptozotocin (60 mg kg-1) (STZ)-induced diabetic and normal rats were studied using the euglycaemic clamp procedure and continuous infusion of somatostatin (1.0 micrograms kg-1 min-1) in pentobarbital-anaesthetized rats. Plasma insulin levels during the 6.0- and 30.0-mU kg-1 min-1 insulin infusions rose to 70-90 microU mL-1 and 500-700 microU mL-1, respectively. Blood glucose concentrations were clamped at 7.5-7.9 mmol L-1 in the diabetic rats and at basal levels or 7.7 mmol L-1 in the non-diabetic (normal) rats. Biosynthetic human C-peptide (0.5 nmol kg-1 min-1) was infused in 12 diabetic and 11 normal rats, resulting in concentrations of 26-41 nmol L-1. The metabolic clearance rate of glucose (MCR) for the diabetic rats receiving C-peptide (12.0 +/- 1.0 mL kg-1 min-1) was significantly (P < 0.01) higher than that in the diabetic rats given saline (6.3 +/- 0.7 mL kg-1 min-1) or a randomly scrambled C-peptide (7.8 +/- 1.3 mL kg-1 min-1) at low-dose insulin infusion but not at the high-dose insulin infusion. In normal rats C-peptide did not significantly increase the MCR for glucose. These results thus demonstrate that C-peptide has the capacity to increase glucose utilization in STZ-induced diabetic rats.

Animals↗

PubMed1996

Expression of the SM22alpha promoter in transgenic mice provides evidence for distinct transcriptional regulatory programs in vascular and visceral smooth muscle cells.

SM22alpha is a putative calcium-binding protein that is expressed in cardiac, smooth, and skeletal muscle lineages during mouse embryogenesis and in adult smooth muscle cells (SMC). To define the mechanisms that regulate smooth muscle-specific gene transcription, we isolated the SM22alpha gene and analyzed its 5'-flanking region for elements that direct smooth muscle expression in transgenic mice. Using a series of promoter deletions, a region of the SM22alpha promoter containing 445 base pairs of 5'-flanking sequence was found to be sufficient to direct the specific expression of a lacZ transgene in mouse embryos in the vascular smooth, cardiac, and skeletal muscle lineages in a temporospatial pattern similar to the endogenous SM22alpha gene. However, in contrast to the endogenous gene, transgene expression was not detected in venous, nor visceral SMCs. This SM22alpha-lacZ transgene was therefore able to distinguish between the transcriptional regulatory programs that control gene expression in vascular and visceral SMCs and revealed heretofore unrecognized differences between these SMC types. These results suggest that distinct transcriptional regulation programs control muscle gene expression in vascular and visceral SMCs.

Animals↗

PubMed1996

Synaptic vesicle recycling in synapsin I knock-out mice.

The synapsins are a family of four neuron-specific phosphoproteins that have been implicated in the regulation of neurotransmitter release. Nevertheless, knock-out mice lacking synapsin Ia and Ib, family members that are major substrates for cAMP and Ca2+/ Calmodulin (CaM)-dependent protein kinases, show limited phenotypic changes when analyzed electrophysiologically (Rosahl, T.W., D. Spillane, M. Missler, J. Herz, D.K. Selig, J.R. Wolff, R.E. Hammer, R.C. Malenka, and T.C. Sudhof. 1995. Nature (Lond.). 375: 488-493; Rosahl, T.W., M. Geppert, D. Spillane, D., J. Herz, R.E. Hammer, R.C. Malenka, and T.C. Sudhof. 1993. Cell. 75:661-670; Li, L., L.S. Chin, O. Shupliakov, L. Brodin, T.S. Sihra, O. Hvalby, V. Jensen, D. Zheng, J.O. McNamara, P. Greengard, and P. Andersen. 1995. Proc. Natl. Acad. Sci. USA. 92:9235-9239; see also Pieribone, V.A., O. Shupliakov, L. Brodin, S. Hilfiker-Rothenfluh, A.J. Czernik, and P. Greengard. 1995. Nature (Lond.). 375:493-497). Here, using the optical tracer FM 1-43, we characterize the details of synaptic vesicle recycling at individual synaptic boutons in hippocampal cell cultures derived from mice lacking synapsin I or wild-type equivalents. These studies show that both the number of vesicles exocytosed during brief action potential trains and the total recycling vesicle pool are significantly reduced in the synapsin I-deficient mice, while the kinetics of endocytosis and synaptic vesicle repriming appear normal.

Animals↗

PubMed1996

Thapsigargin, a weak skin tumor promoter, alters the growth and differentiation of mouse keratinocytes in culture.

Thapsigargin (Tg), applied twice weekly to the backs of CD-1 mice initiated with 7,12-dimethylbenz[a]anthracene, promoted papillomas on the skin of approximately 50% of the mice. Tg alone induced papillomas in 10% of the mice. Although Tg and 12-O-tetradecanoylphorbol-13-acetate (TPA) are synergistic in a keratinocyte co-culture assay for promotion, skin tumor promotion by TPA was inhibited by co-treatment with Tg. Treatment of cultured keratinocytes with non-toxic doses of Tg increased the level of intracellular free Ca(2+)-induced stratification. Tg blocked expression of the spinous layer differentiation marker keratin 1 (K1), while inducing cornification, a marker of differentiation in the granular layer/stratum corneum. In medium with 1.4 mM Ca2+, Tg prolonged keratinocyte proliferation and selected foci of monolayer cells. A Tg-independent cell line, TK-1, was developed from a single dish in which foci continued to expand after Tg removal. Grafting TK-1 cells on to the backs of nude mice as part of a reconstituted skin resulted in the development of dysplastic papillomas with a high rate of progression to squamous cell carcinomas.

Animals↗

PubMed1996

The paralysé mouse mutant: a new animal model of anterior horn motor neuron degeneration.

The survival and morphometric characteristics of lumbar spinal motoneurons were examined in the paralysé mouse mutant. Affected (par/par) mice can be first recognized at approximately postnatal day (PN) 7 to 8 and are characterized by their smaller-than-normal body size, a progressive generalized muscle weakness, and lack of coordination. Mutant mice die by PN16-18, when they have become almost completely paralyzed. Previously, we have shown that this mutation involves alteration of several developmental aspects of the neuromuscular system. However, whether ventral (or anterior) horn motoneurons degenerate and die during the course of the disease was unknown. We report here that at the time the mutant phenotype can be first identified (i.e. approximately PN8), lumbar motoneuron numbers in the lateral motor column of the spinal cord of paralysé mice were not significantly different from those of control littermates. In contrast, by PN14, there was a significant (30 to 35%) decrease in motoneuron numbers in mutant compared to control mice. Furthermore, motoneuron (nuclear and soma) sizes were significantly decreased in the mutants at both stages examined, i.e. PN8 and PN14. These results show that the paralysé mutation involves atrophy and subsequent death of anterior horn motoneurons. Together with the rapid progression and the severity of the disease, these results suggest that the paralysé mouse may represent a good animal model for studying early-onset human motor neuron diseases such as spinal muscular atrophy.

Animals↗

PubMed1996

Development of a fluorescence polarization immunoassay for lorazepam quantification.

Lorazepam concentrations have been quantified in biological fluids using gas chromatography and high-performance liquid chromatography (HPLC). However, these methods are too time consuming and labor intensive for most nonresearch laboratories to offer. A fluorescence polarization immunoassay (FPIA) method for quantification of lorazepam was developed and validated using a reverse-phase HPLC method as the reference method. The FPIA method involves a single liquid-liquid extraction of 100 microliters of either serum or plasma, then direct analysis using the TDxFLx (Abbott Diagnostics, North Chicago, IL). FPIA calibrations were linear between 50 and 800 ng/ml using five calibrators prepared in human serum. Within-run precision (n = 10) for three serum controls (75, 300, and 600 ng/ml) resulted in coefficients of variation (CVs) of 5.7%, 5.4%, and 4.2%, respectively. Between-day precision studies for the three serum controls were 7.0%, 8.9% and 4.9%, respectively (n = 5). The analytical recovery of the three serum controls was 104.9%, 97.3% and 98.3%, respectively. There was an excellent linear correlation between the FPIA and HPLC determinations of 43 patient specimens (r = 0.990, slope = 0.961, intercept = 16.3). No interferences were found from many commonly prescribed nonbenzodiazepine drugs; however, other benzodiazepines that were tested will cross-react with this procedure and give inaccurate results. Therefore, this method should not be used in patients who are receiving other benzodiazepines in addition to lorazepam. The FPIA method described herein can be adapted to reliably measure lorazepam concentrations in serum or plasma.

Anti-Anxiety Agents↗

PubMed1996

Pesticide application and increased dioxin body burden in male and female agricultural workers in China.

Large amounts of the powerful pesticide sodium pentachlorophenol (Na-PCP) salt have been sprayed over vast areas in central China to control schistosomiasis, a parasitic disease of epidemic proportions. Approximately 6000 tons of Na-PCP are produced in China annually. Dioxins, a class of toxic, persistent compounds, are found as impurities in commercial Na-PCP products. These contaminants are released into the environment and significantly contribute to human exposure to dioxins in China. This study was carried out to determine dioxin levels in environmental and human tissue samples from one schistosomiasis area to evaluate the health risks associated with exposure to Na-PCP. Na-PCP pesticide was applied in 1972, 1973, and again in 1978. A total of approximately 454 tons and 902 tons of 5-ppm Na-PCP in water were sprayed over large land and lake problem schistosomiasis areas, respectively. The groups studied were (1) sprayers or handlers of Na-PCP, (2) persons living in the sprayed areas, and (3) persons living in unsprayed areas 300 km north of the sprayed lake located in a city in the Jiangxi province. Individual whole-blood and breast-milk samples were collected and later pooled for dioxin analysis. Also, a sample of commercial Na-PCP was collected. In addition, sediment samples from the lake where Na-PCP was sprayed were collected from four different sites and one control sample was collected from a non-schistosomiasis area. All of the samples were analyzed by high-resolution gas chromatography/mass spectrometry. A sample of Na-PCP used in schistosomiasis regions was analyzed and levels of 2,3,7,8-substituted dibenzodioxin (PCDD) and dibenzofuran (PCDF) congeners were measured. In addition, the international dioxin toxic equivalent (I-TEQ) value of this sample was calculated. Total I-TEQ of 162 parts per billion (ppb) was found in the Chinese Na-PCP product. A pooled breast-milk sample from mothers, female agricultural workers who were born in the schistosomiasis areas where large amounts of Na-PCP were sprayed, had an I-TEQ of 5,4 parts per trillion (ppt), lipid, which was about double that of mothers from control regions, women born in areas not sprayed with Na-PCP (2.6 ppt, lipid). The dioxin I-TEQ values in human blood ranged from 9.0 (subjects 15 to 19 years of age) to 16.3 ppt, lipid (subjects 35 to 70 years of age) in the whole-blood samples from Na-PCP exposed persons, whereas the general population's whole-blood I-TEQs were 4.8 and 6.4 ppt, lipid, respectively. The PCDD/F congener distribution patterns in four sediment samples from schistosomiasis areas were similar to that of Na-PCP. By comparison of specific "fingerprint" congeners (higher chlorinated dioxins and the closely related dibenzofurans) in Na-PCP, human tissues, and sediment samples, we conclude that the chemical pesticide Na-PCP is a source of environmental and human dioxin exposure in the Chinese schistosomiasis area studied. Although human PCDD/F tissue levels in China are low compared with those in more industrialized countries, the elevated I-TEQ levels in exposed persons are cause for concern.

Adolescent↗

PubMed1996

Molecular cloning and photoperiod-regulated expression of gibberellin 20-oxidase from the long-day plant spinach.

Spinach (Spinacia oleracea L.) is a long-day (LD) rosette plant in which stem growth under LD conditions is mediated by gibberellins (GAs). Major control points in spinach are the later steps of sequential oxidation and elimination of C-20 of C20-GAs. Degenerate oligonucleotide primers were used to obtain a polymerase chain reaction product from spinach genomic DNA that has a high homology with GA 20-oxidase cDNAs from Cucurbita maxima L. and Arabidopsis thaliana Heynh. This polymerase chain reaction product was used as a probe to isolate a full-length cDNA clone with an open reading frame encoding a putative 43-kD protein of 374 amino acid residues. When this cDNA clone was expressed in Escherichia coli, the fusion protein catalyzed the biosynthetic sequence GA53-->GA44-->GA19-->GA20 and GA19-->GA17. This establishes that in spinach a single protein catalyzes the oxidation and elimination of C-20. Transfer of spinach plants from short day (SD) to LD conditions caused an increase in the level of all GAs of the early-13-hydroxylation pathway, except GA53, with GA20, GA1, and GA8 showing the largest increases. Northern blot analysis indicated that the level of GA 20-oxidase mRNA was higher in plants in LD than in SD conditions, with highest level of expression in the shoot tips and elongating stems. This expression pattern of GA 20-oxidase is consistent with the different levels of GA20, GA1, and GA8 found in spinach plants grown in SD and LD conditions.

Amino Acid Sequence↗

PubMed1996

Suspension-induced murine keratinocyte differentiation is mediated by calcium.

Modulating extracellular Ca2+ (Cao) and suspension culture are two frequently used methods to induce maturation of cultured human and mouse keratinocytes. To determine if the two methods share a common mechanism, changes in Ca2+ metabolism were studied in suspension cultures of mouse keratinocytes. Spontaneously detached and suspension- cultured keratinocytes in 0.05 microM Ca2+ medium express markers of suprabasal differentiation, while 0.05 microM Ca2+ is not permissive for marker expression by attached keratinocytes. Intracellular free Ca2+ (Cai) increased rapidly after placing keratinocytes in suspension in 0.05 microM Ca2+, reaching levels up to 3- to 4-fold higher than Cai in attached cells after 4-5 h. In suspended cells, the increase in Cai was associated with a 2- to 6- fold increase in Ca2+ transport across plasma membrane as well as depletion of intracellular Ca2+ -stores. Differentiation marker expression and terminal differentiation were inhibited in suspension-cultured keratinocytes by preventing the rise of Cai using either 1,2-bis (o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid to chelate intracellular Ca2+ or ethyleneglycol-bis (beta-aminoethyl ether)- N,N,N',N' -tetraacetic acid to reduce Cao. Together these results indicate that a rise in CAi is a common mechanism controlling differentiation in cultured mouse keratinocytes, and suspension of keratinocytes enhances Ca2+ transport and alters intracellular Ca2+ sequestration producing a rise in Cai.

Animals↗

PubMed1996

Partial trisomy 6p due to maternal t(1;6) translocation.

Partial trisomy 6p with duplications ranging from 6p21 to 6p25 is emerging as an established syndrome. A case of duplication of segment p22-p25 of the short arm of chromosome 6 as the result of a maternal t (1;6)(q44;p22.2) translocation in a mentally retarded girl with congenital anomalies is reported here. The associated phenotypic anomalies are compared with other reported cases of duplication 6p involving adjacent regions.

Adult↗

PubMed1996

In-vitro bronchorelaxing effects of novel nitric oxide donors GEA 3268 and GEA 5145 in guinea-pigs and rats.

Endogenously released nitric oxide (NO) in airways might contribute to physiological bronchodilation; induced production of NO might play a role in the pathogenesis of asthma, although it could also be a compensatory mechanism to other factors that cause bronchoconstriction or inflammation. To investigate the efficacy of NO donors on bronchial tone, the bronchorelaxing efficacies of NO donors, new experimental GEA compounds 3268 and 5145 (oxatriazole sulphonylamides) were compared with those of sodium nitroprusside and SIN-1 (3-morpholinosydnonimine) and to the standard beta 2-adrenergic agonist, salbutamol, in bronchi of guinea-pigs and rats in-vitro. Their relaxing effects were also studied in rat mesentery arteries to compare the selectivity for airways. The capacity of the NO donors to produce nitrites and nitrates was assayed by the Griess reaction. The novel NO donors GEA 3268 and GEA 5145 were more potent bronchorelaxing agents than the old NO donors sodium nitroprusside and SIN-1. In guinea-pig bronchi, however, salbutamol was most potent. In rat bronchi the GEA compounds induced the strongest relaxation effect when compared with the old NO donors or with salbutamol. The airway selectivity of the drugs studied decreased in the order of salbutamol, SIN-1, GEA 5145, GEA 3268, sodium nitroprusside. The nitrites and nitrates produced spontaneously did not correlate with the efficacy of the relaxants. The results obtained suggest that NO is only partly responsible for the relaxation and the potency is dependent on the animal species and constricting agents used.

Albuterol↗

PubMed1996

Effects of potassium-channel opener HOE 234 in guinea-pig airways.

The mechanism of the bronchospasmolytic effect of HOE 234 ((3S,4R)-3-hydroxy-2,2-dimethyl-4-(2-oxo-1-pyrrolidinyl) 6-phenylsulphonylchromane hemihydrate), a novel opener of ATP-sensitive potassium channels, has been studied by in-vitro testing in ring preparations of trachea and different parts of the principle bronchus of guinea-pigs, using methacholine, histamine and KCl as preconstrictors. The contribution of prostanoids was estimated in the presence and absence of indomethacin. Regional differences in the bronchodilatory effect of HOE 234 were compared with that of salbutamol. HOE 234 had a concentration-dependent relaxing effect on the smooth muscle preparations. In the absence of indomethacin no regional differences were seen for the effect of HOE 234 against the metacholine or KCl preconstriction, whereas with histamine a particularly strong relaxation was detectable in trachea. In the presence of indomethacin, distal bronchus after methacholine and trachea after KCl preconstriction were relaxed significantly more strongly than the other parts. The relaxation of the histamine-constricted trachea rings was not increased in comparison with that without indomethacin pretreatment. Thus the bronchospasmolytic effect of HOE 234 varied depending on the pretreatment, method of preconstriction and part of the airways examined. It was strongest in trachea rings with histamine preconstriction but its potency was clearly less than that of salbutamol. The latter had regionally different effects, being stronger in trachea than in the bronchi for all methods of preconstriction. The results suggest that the formation of bronchoconstricting prostanoids can attenuate parts of the relaxing effect of HOE 234. The possible advantages of HOE 234 as an anti-asthma drug might be related to an effect on bronchial hyper-reactivity and mucus secretion, because as a direct bronchodilator it is inferior to salbutamol.

Adrenergic beta-Agonists↗

PubMed1996

Position-dependent ATT initiation during plant pararetrovirus rice tungro bacilliform virus translation.

The expression of the rice tungro bacilliform virus open reading frame I was studied in transiently transfected protoplasts. Expression occurs despite the presence of a long leader sequence and the absence of a proper ATG initiation codon. Translation is initiated at an ATT codon. The efficiency of initiation in rice protoplasts depends strongly on the mechanism by which ribosomes reach this codon. From the effects of scanning-inhibiting structures inserted into different leader regions, it can be deduced that this mechanism is related to the ribosome shunt described for cauliflower mosaic virus 35S RNA. The process delivers initiation-competent ribosomes to the region downstream of the leader and is so precise that only the second of two potential start codons only 12 nucleotides apart is recognized. The ATT codon that is used when it is present downstream of the leader is hardly recognized as a start codon by ribosomes that reach it by scanning.

Base Sequence↗

PubMed1996

Drosophila homologs of the proto-oncogene product PEBP2/CBF beta regulate the DNA-binding properties of Runt.

The Drosophila runt gene is the founding member of the Runt domain family of transcriptional regulators. Mammalian Runt domain genes encode the alpha subunit of the heterometric DNA-binding factor PEBP2/CBF. The unrelated PEBP2/CBF beta protein interacts with the Runt domain to increase its affinity for DNA. The conserved ability of the Drosophila Runt protein to respond to the stimulating effect of mammalian PEBP2/CBF beta indicated that flies were likely to have a homologous beta protein. Using the yeast two-hybrid system to isolate cDNAs for Runt-interacting proteins, we identified two Drosophila genes, referred to as Brother and Big-brother, that have substantial sequence homology with PEBP2/CBF beta. Yeast two-hybrid experiments as well as in vitro DNA-binding studies confirmed the functional homology of the Brother, Big-brother, and PEBP2/CBF beta proteins and demonstrated that the conserved regions of the Runt and Brother proteins are required for their heterodimeric interaction. The DNA-bending properties of Runt domain proteins in the presence and absence of their partners were also examined. Our results show that Runt domain proteins bend DNA and that this bending is influenced by Brother protein family members, supporting the idea that heterodimerization is associated with a conformational change in the Runt domain. Analysis of expression patterns in Drosophila embryos revealed that Brother and Big-brother are likely to interact with runt in vivo and further suggested that the activity of these proteins is not restricted to their interaction with Runt.

Amino Acid Sequence↗

PubMed1996

Modulation of thermal induction of hsp70 expression by Ku autoantigen or its individual subunits.

Previously, we proposed a dual control mechanism for the regulation of the heat shock response in mammalian cells: a positive control mediated by the heat shock transcription factor HSF1 and a negative control mediated by the constitutive heat shock element-binding factor (CHBF). To study the physiological role of CHBF in the regulation of heat shock response, we purified CHBF to apparent homogeneity and showed it to be identical to the Ku autoantigen, a heterodimer consisting of 70-kDa (Ku-70) and 86-kDa (Ku-80) polypeptides. To study further the functional significance of Ku/CHBF in the cellular response to heat shock, we established rodent cell lines that stably and constitutively overexpressed one or both subunits of the human Ku protein, and examined the thermal induction of hsp70 and other heat shock proteins in these Ku-overexpressing ing cells. We show that expression of the human Ku-70 and Ku-80 subunits jointly or of the Ku-70 subunit alone specifically inhibits heat-induced hsp70 expression. Conversely, expression of human Ku-80 alone does not have this effect. Thermal induction of other heat shock proteins in all of the Ku-overexpressing cell lines appears not to be significantly affected, nor is the state of phosphorylation or the DNA-binding ability of HSF1 affected. These findings support a model in which hsp70 expression is controlled by a second regulatory factor in addition to the positive activation of HSF1. The Ku protein, specifically the Ku-70 subunit, is involved in the regulation of hsp70 gene expression.

Animals↗

PubMed1996

Agonist- and CPA-induced elevation of cytoplasmic free Ca2+ in intact valvular endothelium from rabbits.

Intracellular Ca2+ signals of intact endothelium from rabbit aortic or pulmonic valves loaded with fura 2 were studied using imaging fluorescence microscopy. Agonists such as ATP or carbachol and inhibitors of endoplasmic reticulum (ER) Ca(2+)-ATPase such as cyclopiazonic acid (CPA), thapsigargin, and 2', 5'-di(tert-butyl)-1, 4,-benzohydroquinone (BHQ) induced an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) that was maintained above the prestimulated levels as long as extracellular Ca2+ was present, indicating that the maintained [Ca2+]i increase is dependent on the entry of extracellular Ca2+. The voltage-gated channel was not found to contribute to [Ca2+]i increase. SK&F-96365, a receptor-operated cation channel (ROC) blocker, and 2-nitro-4-carboxyphenyl-N, N-diphenylcarbamate (NCDC), a postulated phospholipase C inhibitor, were shown to effectively block ROC, since they greatly reduced the maintained [Ca2+]i increase caused by ATP, but not that caused by CPA, which was blocked by Ni2+. To further investigate the Ca2+ influx involved in this process, divalent cation entry was measured as Mn2+ quenching of fura 2 fluorescence at 360-nm excitation wavelength. At rest, fluorescence quenching at 360 nm by Mn2+ was observed, which was inhibited by Ni2+ but not by NCDC, indicating Mn2+ entry through the leak pathway. The quenching was enhanced following ATP stimulation, and this enhancement was abolished by pretreatment with NCDC. In contrast, the rate of Mn2+ quenching was unaffected by the application of CPA. These results demonstrate that ATP stimulates divalent cation influx that is not related to the Ca2+ content of ER. Abolition of Ca2+ uptake into ER was postulated to increase the effectiveness of the Ca2+ leak in raising [Ca2+]i.

Adenosine Triphosphate↗

PubMed1996

An endogenous ATP-sensitive glutathione S-conjugate efflux mechanism in Xenopus laevis oocytes.

Constitutive efflux mechanisms for reduced glutathione (GSH) and the glutathione S-conjugates S-ethylglutathione (ethyl-SG) and S-(2,4-dinitrophenol)-glutathione (DNP-SG) were examined in Xenopus laevis oocytes. Oocytes were loaded by either microinjection with 50 nl of the 3H-labeled compounds or were exposed to unlabeled 1-chloro-2,4-dinitrobenzene and efflux of DNP-SG synthesized within the oocytes measured spectrophotometrically. Efflux of unlabeled DNP-SG (approximately 1.2 mM intracellular concentration) and microinjected 0.5 mM [3H]DNP-SG was a linear function of time, with approximately 20% released in 3 h at 25 degrees C. [3H] ethyl-SG, 0.5 mM, was released at a comparable rate, whereas only 4% of a tracer dose of [3H]GSH (2.5 mM intracellular GSH) was released in 3 h. Efflux of all three compounds was temperature sensitive and inhibited after ATP depletion but unaffected when Na+ in the culture medium was replaced with K+ or when the pH was changed from 7.5 to either 6.8 or 8.0. Efflux was saturable, with apparent Michaelis constant values of 0.91 +/- 0.19, 0.44 +/- 0.25, and 5.3 +/- 2.2 mM for DNP-SG, ethyl-SG, and GSH, respectively. Bilirubin ditaurate, 0.5 mM, cis-inhibited efflux of 0.5 mM [3H]DNP-SG, 0.5 mM [3H]ethyl-SG, and 2.5 mM [3H]GSH. DNP-SG and ethyl-SG efflux was also cis-inhibited by other glutathione S-conjugates, 0.25 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, 0.5 mM sulfobromophthalein, and 0.5 mM dibromosulfophthalin, but not by 0.25 mM taurocholate. [3H]GSH release (2.5 mM) was unaffected by these compounds or by 10 mM extracellular GSH or methionine. These findings indicate that Xenopus oocytes have an endogenous ATP-sensitive mechanism for extruding glutathione S-conjugates, with properties comparable to ATP-dependent glutathione S-conjugate/organic anion transport systems described in a variety of cell types. However, in contrast to mammalian cells, GSH and ethyl-SG release from Xenopus oocytes was also inactivated after cellular ATP depletion but was not sensitive to membrane depolarization in high-K+ medium or trans-stimulated by extracellular GSH, indicating that efflux of these organic anions from Xenopus laevis oocytes is also mediated by an ATP-sensitive mechanism.

Adenosine Triphosphate↗

PubMed1996

Polyol pathway and osmoregulation in JS1 Schwann cells grown in hyperglycemic and hyperosmotic conditions.

To test the osmoregulatory rules of Schwann cell aldose reductase (AR) and myo-inositol, JS1 Schwann cells were grown under control and hyperosmotic conditions with and without excess glucose or galactose. JS1 cells cultured in control conditions possessed AR protein and activity that were not altered by the inclusion of 25 mM glucose or galactose. Following culture with 100 mM NaCl, there was a decline in cell number accompanied by an increase in AR activity, both of which were attenuated by the addition of 25 mM glucose or galactose. Sorbitol was not detected in JS1 Schwann cells following culture in control, glucose-supplemented, or hyperosmotic medium, and dulcitol accumulated only following culture with galactose. However, both polyols were dramatically increased in JS1 cells cultured in hyperosmotic medium supplemented with 25 mM glucose or galactose. In contrast, myo-inositol levels were elevated only during hyperosmotic exposure but decreased when glucose or galactose was also present. These data are consistent with the use of polyol formation by JS1 Schwann cells as a means of responding to osmotic stress.

Aldehyde Reductase↗