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41.
A Celis P Madsen H V Nielsen H H Rasmussen H Thiessen J B Lauridsen B van Deurs J E Celis 《FEBS letters》1988,227(1):14-20
A mouse monoclonal antibody (mAB 22-II-D8B) raised against lysed transformed human amnion cells (AMA) has been characterized. The mAB decorated the Golgi apparatus in growing and quiescent cultured monolayer cells (fibroblasts and epithelial cells) of various species as determined by double immunofluorescence labeling and colocalization with galactosyltransferase antibodies. It reacted with the acidic human proteins IEF 58 (Mr = 29,000) and 57a, respectively (Mr = 30,000) (HeLa protein catalogue number; [(1982) Clin. Chem. 28, 766]), Golgi staining was also observed in BS-C-1 cells microinjected with mAB 22-II-D8B suggesting that the epitopes recognized by the antibody are most likely located on the cytoplasmic face of the membranes. The precise localization of the antigens to the various cisternae of the Golgi apparatus could not be demonstrated by immunogold cytochemistry on ultrathin cryosections due to either weak reactivity of the antibody or low concentration of the antigens. Immunofluorescence staining with mAB 22-II-D8B of lymphoid human Molt-4 cells and some human tissues failed to reveal any significant staining even though these expressed high levels of both IEF 58 and 57a. These results are taken to imply that the epitopes recognized by mAB 22-II-D8B may be masked in some cell types. 相似文献
42.
Radiation inactivation is a method to determine the apparent target size of molecules. In this report we examined whether radiation inactivation of various enzymes and brain receptors is influenced by the preparation of samples preceding irradiation. The apparent target sizes of endogenous acetylcholinesterase and pyruvate kinase from rat brain and from rabbit muscle and benzodiazepine receptor from rat brain were investigated in some detail. In addition the target sizes of alcohol dehydrogenase (from yeast and horse liver), beta-galactosidase (from Escherichia coli), lactate dehydrogenase (endogenous from rat brain), and 5-HT2 receptors, acetylcholine muscarine receptors, and [35S] butyl bicyclophosphorothionate tertiary binding sites from rat brain were determined. The results show that apparent target sizes are highly influenced by the procedure applied for sample preparation before irradiation. The data indicate that irradiation of frozen whole tissue as opposed to lyophilized tissue or frozen tissue homogenates will estimate the smallest and most relevant functional target size of a receptor or an enzyme. 相似文献
43.
Niels Peter Revsbech Lars Peter Nielsen Peter Bondo Christensen Jan Srensen 《Applied microbiology》1988,54(9):2245-2249
The construction of a microsensor which can be used to measure O2 and N2O simultaneously is described. The microsensor exhibited a linear response to both O2 and N2O, and the response to N2O was independent of the O2 concentration and vice versa. The N2O detection limit of a microsensor with a tip diameter of 20 μm was around 1 μmol liter−1. The signals for O2 and N2O were affected by hydrogen sulfide, but other interfering agents were not observed in the biofilms and sediments analyzed. Microprofiles of O2 and N2O were measured in a biofilm which was exposed to acetylene to block the N2O reductase activity of denitrifying bacteria. O2 penetrated about 0.5 mm into the biofilm and was not affected by acetylene, but the N2O concentration at 1.4 mm depth increased from 32 to 411 μmol liter−1 after the addition of the inhibitor. The shape of the N2O profile after the addition of acetylene showed that denitrification (denitrifying activity) was detectable in all anoxic layers of the biofilm. 相似文献
44.
J Vervoort W J van Berkel S G Mayhew F Müller A Bacher P Nielsen J LeGall 《European journal of biochemistry》1986,161(3):749-756
Megasphaera elsdenii and Desulfovibrio vulgaris apoflavodoxins have been reconstituted with riboflavin 3',5'-bisphosphate. Several biochemical and biophysical properties of the complexes have been investigated and the results are compared with the properties of the native proteins. The dissociation constant of the modified complex of M. elsdenii flavodoxin is increased by a factor of about 23 by comparison with that of the native protein. The rate constant for the formation of the complex of M. elsdenii flavodoxin is about 26 times lower than that for the native protein. The redox potential of the transition between the oxidized and semiquinone state is similar to that of the native protein. On the other hand, the redox potential of the semiquinone-hydroquinone transition is about 20 mV more negative than that of the native protein. Absorbance and circular dichroic spectra of the protein-bound artificial prosthetic group and the protein-bound natural prosthetic group are very similar. In both the oxidized and in the fully reduced state only minor differences in interaction between the isoalloxazine ring and the apoprotein for the two flavin derivatives are found by 13C and 15N NMR. 31P-NMR studies show that the 5'-phosphate group of the two flavin derivatives is bound in the same way and that it is dianionic in the complex. In contrast, the 3'-phosphate group in riboflavin 3',5'-bisphosphate is monoanionic or even neutral when bound to the protein. The 3'-phosphate group is also close to or on the surface of the protein. Desulfovibrio vulgaris apoflavodoxin has an affinity for riboflavin 3',5'-bisphosphate which is 10 times higher as compared to Megasphaera elsdenii apoflavodoxin (Ka = 10(8) M-1). Also the association rate constant of Desulfovibrio vulgaris apoprotein and riboflavin 3'5'-bisphosphate is found to be 10 times faster than for the Megasphaera elsdenii flavodoxin reaction. The dissociation behaviour of native Desulfovibrio vulgaris flavodoxin measured under identical conditions as for the riboflavin 3',5'-bisphosphate analog gives a value (Kd approximately equal to 0.2 nM) which is considerably lower than reported earlier [Dubourdieu, M., MacKnight, M. L. & Tollin, G. (1974) Biochem. Biophys. Res. Commun. 60, 649-655]. The results are discussed in the light of the existing crystallographic data of flavodoxins and the recently proposed theory on the regulation of the redox potential in flavoproteins [Moonen, C. T. W., Vervoort, J. & Müller, F. (1984) in Flavins and flavoproteins, pp. 493-496, Walter de Gruyter, Berlin]. 相似文献
45.
Monoamine Neurotransmitter Metabolism and Locomotor Activity During Chemical Hypoxia 总被引:4,自引:1,他引:3
The effects of hypoxia on metabolism of 5-hydroxytryptamine (5-HT or serotonin) and 3,4-dihydroxyphenylethylamine (DA or dopamine) were compared with those on open-field activity in male CD-1 mice. Chemical hypoxia was induced with NaNO2. Hypoxia did not alter striatal concentrations of DA, 5HT, Trp, Tyr, 5-hydroxyindoleacetic acid, or homovanillic acid. However, NaNO2 (75 mg/kg) reduced the rates of conversion of [3H]Tyr to [3H]DA (-41%) and [3H]Trp to [3H]5-HT (-39%). Hypoxia also reduced dihydroxyphenylacetic acid (DOPAC) levels (-27%) and DOPAC/DA ratios (-20%). Open-field behavior, as measured in an automated activity monitor, decreased in a dose-dependent fashion with 75-150 mg/kg of NaNO2 (-35 to -90%). Comparison with previous studies suggests that the syntheses of dopamine, serotonin, and the amino acids are equally vulnerable to hypoxic insults but may be less sensitive than the synthesis of acetylcholine. 相似文献
46.
Serum steroid levels in intact and endocrine ablated BALB/c nude mice and their intact littermates 总被引:2,自引:0,他引:2
N Brünner B Svenstrup M Spang-Thomsen P Bennett A Nielsen J Nielsen 《Journal of steroid biochemistry》1986,25(3):429-432
An investigation was made of the serum steroid levels found in intact and endocrine ablated nude mice of both sexes and in their intact homozygous littermates. The results showed that nude mice have a normal steroidogenesis, but with decreased levels of circulating steroids compared to those of the littermates. The efficacy of the endocrine ablations was confirmed by the reduction in serum oestrone following oophorectomy, and by the reduction in serum testosterone and progesterone following orchiectomy. The normal steroidogenesis in nude mice, and the similarities between mouse and man with regard to changes in serum steroids following oophorectomy and orchiectomy, support the usefulness of human tumor xenograft models for the study of hormone-tumor interactions. 相似文献
47.
48.
Biosynthesis of riboflavin. Enzymatic formation of 6,7-dimethyl-8-ribityllumazine from pentose phosphates 总被引:2,自引:0,他引:2
P Nielsen G Neuberger I Fujii D H Bown P J Keller H G Floss A Bacher 《The Journal of biological chemistry》1986,261(8):3661-3669
The xylene ring of riboflavin originates by dismutation of the precursor, 6,7-dimethyl-8-ribityllumazine. The formation of the latter compound requires a 4-carbon unit as the precursor of carbon atoms 6 alpha, 6, 7, and 7 alpha of the pyrazine ring. The formation of riboflavin from GTP and ribose phosphate by cell extract from Candida guilliermondii has been observed by Logvinenko et al. (Logvinenko, E. M., Shavlovsky, G. M., Zakal'sky, A. E., and Zakhodylo, I. V. (1982) Biokhimiya 47, 931-936). We have studied this enzyme reaction in closer detail using carbohydrate phosphates as substrates and synthetic 5-amino-6-ribitylamino-2,4-(1H,3H)-pyrimidinedione or its 5'-phosphate as cosubstrates. Several pentose phosphates and pentulose phosphates can serve as substrate for the formation of riboflavin with similar efficiency. The reaction requires Mg2+. Various samples of ribulose phosphate labeled with 14C or 13C have been prepared and used as enzyme substrates. Radioactivity was efficiently incorporated into riboflavin from [1-14C]ribulose phosphate, [3,5-14C]ribulose phosphate, and [5-14C]ribulose phosphate, but not from [4-14C]ribulose phosphate. Label from [1-13C]ribose 5-phosphate was incorporated into C6 and C8 alpha of riboflavin. [2,3,5-13C]Ribose 5-phosphate yielded riboflavin containing two contiguously labeled segments of three carbon atoms, namely 5a, 9a, 9 and 8, 7, 7 alpha. 5-Amino-6-[1'-14C] ribitylamino-2,4 (1H,3H)-pyrimidinedione transferred radioactivity exclusively to the ribityl side chain of riboflavin in the enzymatic reaction. It follows that the 4-carbon unit used for the biosynthesis of 6,7-dimethyl-8-ribityllumazine consists of the pentose carbon atoms 1, 2, 3, and 5 in agreement with earlier in vivo studies. 相似文献
49.
Smith C. A.; Bisgard G. E.; Nielsen A. M.; Daristotle L.; Kressin N. A.; Forster H. V.; Dempsey J. A. 《Journal of applied physiology》1986,60(3):1003-1010
We have compared the ventilatory responses of intact and carotid body-denervated (CBD) goats to moderate [partial pressure of O2 in arterial blood; (Pao2) approximately 44 Torr] and severe (Pao2 approximately 33 Torr) many time points for up to 7 days of hypobaria. In the intact group there were significant time-dependent decreases in partial pressure of CO2 in arterial blood (PaCO2) in both moderate and severe hypoxemia (approximately-7 and -11 Torr) that were largely complete by 8 h of hypoxemia and maintained throughout. Acute restoration of normoxia in chronically hypoxic intact animals produced time-dependent increases in Paco2 over 2 h, but hypocapnia persisted relative to sea-level control. Arterial plasma [HCO3-] and [H+] decreased, and [Cl-] increased with a time course and magnitude consistent with developing hypocapnia. Chronic CBD, per se, resulted in a sustained, partially compensated respiratory acidosis, as PaCO2 rose 6 Torr and base excess rose 3 mEq/1, [Cl-] fell 1 mEq/1, and pHa fell 0.01 units. During exposure to identical levels of arterial hypoxemia as in the intact group. CBD animals showed no significant changes in PaCO2, [H+]a, or [HCO3-]a at any time during moderate or severe hypoxemia. Plasma [C1-] remained within the normal range throughout exposure to moderate hypoxia and increased in severe hypoxia. In a few instances some hypocapnia was observed, but this was highly inconsistent and was always less than one-third of that observed in intact goats. In contrast to intact goats, acute restorations of normoxia in the chronically hypoxic CBD goats always caused hyperventilation.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
50.
We have investigated the subunit structure of Ca2+-transport ATPase in human erythrocyte membranes using radiation inactivation analysis. All inactivation data were linear on a semilog plot down to at least 20% of the control activity. We found a target size for the calmodulin-dependent Ca2+-ATPase activity of 331 kDa, consistent with the presence of this enzyme as a dimer in calmodulin-depleted ghosts. Membranes which had been saturated with calmodulin before irradiation yield a a similar size of 317 kDa, implying that activation of Ca2+-transport ATPase by calmodulin does not involve significant change in oligomeric structure. Basal (calmodulin-independent) Ca2+-ATPase activity corresponded to a size of 290 kDa, suggesting that this activity resides in the same, or similar-sized, complex as the calmodulin-dependent activity. Mg2+-ATPase activity, however, was found to reside in a smaller complex of 224 kDa, which proved to be statistically distinct from the target size of Ca2+-ATPase activity. It would appear that Mg2+-ATPase is a distinct entity whose function is likely unrelated to the Ca2+-transport ATPase. 相似文献