Microsomal-catalyzed hydroperoxide-dependent C-oxidation of amines

Organic hydroperoxides are capable of supporting the C-oxidation of several different amines in the presence of hepatic microsomes. Evidence is presented that indicates that microsomal cytochrome P-450 acts as the catalyst. Removal of the NADPH-cytochrome $\text{c}$ oxidoreductase or essential phospholipid from microsomes does not significantly affect the peroxidase activity. Of the amine substrates C-oxidized by organic hydroperoxides in the presence of microsomes, only aminopyrine and dimethylaniline are rapidly oxidized by hydroperoxides in the presence of catalase. The catalase-mediated reaction can also be distinguished from the microsomal-catalyzed reaction by the use of differential inhibitors.     

Classical plant taxonomic ambiguities extend to the molecular level

Summary The molecular evolution of cytochrome c from angiosperms is compared to that from vertebrates. On the basis of a cladistic analysis from 26 plant species, compared to that from 27 vertebrate species, we find that although the vertebrate sequences yield reasonably well-defined minimal trees that are congruent with the biological tree, the plant sequences yield multiple minimal trees that are not only highly incongruent with each other, but none of which is congruent with any reasonably biological tree. That is, the plant sequence set is much more homoplastic than that of the animal. However, as judged by the relative rate test, the extent of divergence, and degree of functional constraint, cytochrome c evolution in plants does not appear to differ from that of vertebrates.     

Influence of single amino acids on the development of hamster one-cell embryos in vitro

One-cell hamster embryos placed in culture have always shown a complete block to development at the two-cell stage. In a preliminary study using a chemically defined culture medium containing 20 amino acids (HECM-1), many one-cell embryos were able to escape the "two-cell block" and develop to the four-cell stage. Use of a simpler formulation containing only the amino acids hypotaurine and glutamine revealed marked inhibitory and stimulatory effects of adding the other amino acids. In the first experiment, 19 amino acids were separately examined for effects on one-cell embryo development. Six amino acids (phenylalanine, valine, isoleucine, tyrosine, tryptophan, and arginine) inhibited embryo development (reduced mean cell number; MCN), and three others (glycine, cystine, and lysine) stimulated development (increased MCN), compared with basic medium containing only glutamine and hypotaurine (low control). When the responses with the six inhibitory amino acids were totalled, only 3 of 185 (2%) one-cell embryos reached the six-or seven-cell stage compared to a total of 15 of 76 (20%) embryos that developed to these stages using the three stimulatory amino acids. When tested together in a second experiment, the six inhibitory amino acids significantly reduced the MCN, from 4.28 +/- 0.44 (low control) to 3.71 +/- 0.55. In this group, 17 of 117 (15%) of one-cell embryos reached more than four-cell and only 4 of 117 (3%) reached six- or 7-cell stages, compared with 39 of 117 (33%) and 12 of 117 (10%), respectively, for the basal medium group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental and Regional Expression of NMDA Receptor Subtypes Containing the NR2D Subunit in Rat Brain

Abstract: The regional and developmental expression of NMDA receptors containing the NR2D subunit was analyzed on the level of the subunit mRNA and protein in rat brain. RNase protection experiments indicated that among two proposed splice variants of the NR2D subunit, only the NR2D-2 subunit is expressed. The regional distribution of the NR2D subunit protein was visualized with a newly developed NR2D-2 subunit-specific antiserum on brain sections using the histoblot technique. In adult brain, NR2D immunoreactivity was mainly restricted to diencephalic, mesencephalic, and brainstem structures. During postnatal development, the NR2D subunit was detected transiently in certain regions, such as the ventro-basal complex of the thalamus, hippocampus, inferior colliculus, and brainstem reticular formation, suggesting that NR2D subunit-containing receptors play a role in these brain areas only during development. The level of NR2D subunit mRNA and protein decreased during late postnatal development. However, significant levels of NR2D subunit mRNA and protein were present in adulthood, in particular, in the globus pallidus, thalamus, subthalamic nuclei, and superior colliculus. These results indicate a functional relevance for NMDA receptors containing the NR2D subunit in the developing and adult brain, although its expression in the adult brain is less prominent and restricted to a few brain areas.     

HCO3− and CO2 leakage from Synechococcus UTEX 625

The leakage of various inorganic carbon species from air-grown cells of Synechococcus UTEX 625 was investigated after a light to dark transition or during a light period using a mass spectrometer under a wide variety of experimental conditions. Total inorganic carbon efflux and CO₂ efflux during the initial period of darkness were measured with or without carbonic anhydrase in the reaction medium respectively. The HCO₃^? efflux after a light to dark transition was estimated by difference. Carbon dioxide efflux in the light was measured by inhibiting CO₂ transport with either Na₂S or COS₃ or quenching the ¹³C inorganic carbon transport by the addition of ¹²C inorganic carbon in excess. In cells in which CO₂ fixation was inhibited, when only the HCO₃^? transport system was fully operative, CO₂ effluxed continuously during the light period at a rate equal to about 25% of that in darkness. When only the CO₂ transport system was operative, HCO₃^? effluxed during the light period. The difference between the light and dark efflux rates was consistent with a 0.6 unit decrease in the intracellular pH upon darkening the cells. The permeabilities of the cell for CO₂ (2.94 ± 0.14 ± 10^?8ms^?1; mean ± SE, n=137) and HCO₃^? (1.4–1.7 ± 10^?9 ms^?1) were calculated.     

细胞色素c在氨基酸及二肽修饰金电极上的电化学反应

系统地研究了细胞色素ｃ在多种氨基酸和多肽修饰电极上的电化学反应。并对影响加速细胞色素ｃ电化学反应的因素进行了讨论。     

Autoradiographic Distribution and Characteristics of High- and Low-Affinity Polyamine-Sensitive [3H]Ifenprodil Sites in the Rat Brain: Possible Relationship to NMDAR2B Receptors and Calmodulin

Abstract: We have studied the regional distribution and characteristics of polyamine-sensitive [³H]ifenprodil binding sites by quantitative autoradiography in the rat brain. In forebrain areas ifenprodil displaced [³H]ifenprodil (40 nM) in a biphasic manner with IC₅₀ values ranging from 42 to 352 nM and 401 to 974 µM. In hindbrain regions, including the cerebellum, ifenprodil displacement curves were monophasic with IC₅₀ values in the high micromolar range. Wiping studies using forebrain slices (containing both high- and low-affinity sites) or cerebellar slices (containing only the low-affinity site) showed that high- and low-affinity ifenprodil sites are sensitive to spermine and spermidine, to the aminoglycoside antibiotics neomycin, gentamicin, and kanamycin, and to zinc. Two calmodulin antagonists, W7 and calmidazolium, also displaced [³H]ifenprodil from both sites. Other calmodulin antagonists, including trifluoperazine, prenylamine, and chlorpromazine, selectively displaced [³H]ifenprodil from its low-affinity site in hindbrain and forebrain regions. High-affinity [³H]ifenprodil sites, defined either by ifenprodil displacement curves or by [³H]ifenprodil binding in the presence of 1 mM trifluoperazine, were concentrated in the cortex, hippocampus, striatum, and thalamus with little or no labeling of hindbrain or cerebellar regions. This distribution matches that of NMDAR2B mRNA, supporting data showing that ifenprodil has a preferential action at NMDA receptors containing this subunit. Low-affinity [³H]ifenprodil sites have a more ubiquitous distribution but are especially concentrated in the molecular layer of the cerebellum. [³H]Ifenprodil was found to bind to calmodulin-agarose with very low affinity (IC₅₀ of ifenprodil = 516 µM). This binding was displaced by calmodulin antagonists and by polyamines, with a potency that matched their displacement of [³H]ifenprodil from its low-affinity site in brain sections. However, the localization of the low-affinity [³H]ifenprodil site does not strictly correspond to that of calmodulin, and its identity remains to be further characterized. The restricted localization of high-affinity [³H]ifenprodil binding sites to regions rich in NMDAR2B subunit mRNA may explain the atypical nature of this NMDA antagonist.     

Activity of the tetramer and octamer of glutamine synthetase isoforms during primary leaf ontogeny of sugar beet (Beta vulgaris L.)

In extracts from the primary leaf blade of sugar beet (Beta vulgaris L.) we separated a chloroplastic isoform (GS 2) of glutamine synthetase (GS, EC 6.3.1.2) and one or two (depending on leaf age) cytosolic isoforms (GS 1a and GS 1b). The latter were prominent in the early (GS 1a) and late stages of leaf ontogeny (GS 1a and GS 1b), whereas during leaf maturation GS 2 was the predominantly active GS isoform. The GS 1 isoforms were active exclusively in the octameric state although tetrameric GS 1 protein was detected immunologically. Their activity stayed at a relatively constant level during leaf ontogeny; an increase was observed only in the senescent leaf. The activity of GS 2, however, changed drastically during primary leaf ontogeny and was modulated by changes in the oligomeric state of the active enzyme. In the early and late stages of leaf ontogeny when GS 2 activity was low (lower than that of the GS 1 isoforms), GS 2 was active only in the octameric state. In the maturing leaf, when GS 2 activity had reached its maximum level (much higher than that of the GS 1 isoforms), 80 of total GS 2 activity was due the activity of the tetrameric form of the enzyme and 20 was due to octameric GS 2. Tetrameric GS 2 was a hetero-tetramer and thus not the unspecific dissociation product of homo-octameric GS 2. In addition, GS 2 activity was modulated by an activation/inactivation of the tetrameric GS 2 protein. Due to an activation of the GS 2 tetramer, the activity of tetrameric GS 2 increased during leaf maturation from zero level 23-fold compared with that of GS 1a and 18-fold compared with that of GS 1b. Possible activators of tetrameric GS 2 are thiol-reactive substances. During leaf senescence, GS 2 activity decreased to zero; this decrease was due to an inactivation of the tetrameric GS 2 protein probably caused by oxidation.Abbreviations FLL final lamina length - FPLC fast protein liquid chromatography - GS glutamine synthetase - GHA -glutamyl hydroxamate - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase Dr. Roger Wallsgrove's (Rothamsted Experimental Station, Harpenden, UK) generous gift of GS antiserum is greatly appreciated.