Involvement of the cystathionine pathway in the biosynthesis of glutathione by isolated rat hepatocytes

The ability of l-methionine to support glutathione biosynthesis has been investigated in isolated rat hepatocytes under conditions of normal and depleted glutathione status. The addition of l-[³⁵S]methionine or [l-[³⁵S]homocysteine to incubation media containing hepatocytes results in the incorporation of ³⁵S into intracellular glutathione. Additionally both l-methionine and l-homocysteine are capable of supporting the resynthesis of glutathione in isolated hepatocytes after prior depletion with diethyl maleate. The inclusion in the incubation medium of 1 mm propargylglycine, which is an irreversible inhibitor of the terminal enzyme of the cystathionine pathway, substantially blocks the incorporation of ³⁵S from methionine and l-homocysteine into cellular glutathione. Propargylglycine treatment of hepatocytes in the presence of [³⁵S]methionine is shown to result in the intracellular accumulation of [³⁵S]cystathionine. These results strongly support the conclusion that in rat hepatocytes the cystathionine pathway enables methionine to provide a significant source of l-cysteine for the support of glutathione biosynthesis, under both normal and glutathione-depleted conditions.     

Carboranyl peptide-antibody conjugates for neutron-capture therapy: preparation, characterization, and in vivo evaluation.

Two model peptides rich in boron and prepared by Merrifield syntheses, dansyl.(nido-CB)2, (1) and dansyl.(nido-CB)10.Lys.Ac (2), where nido-CB represents the alpha-amino acid [nido-7-CH3-8-(CH2)3CH-(NH2)COOH-7,8-C2B9H10]-, were conjugated with the anti-CEA mAb T84.66 using peptide active ester reagents. The dansyl groups provided a means of fluorimetric analysis of mAb conjugates which was augmented by conventional amino acid analyses for nido-CB. The conjugate of 1 contained an average of 63 B atoms per mAb molecule. The mAb conjugate of 2 was chromatographically separated into a strongly fluorescent high molecular weight aggregated fraction (HMW) and a less intensely fluorescent monomeric fraction. Both fractions retained immunoreactivity. The HMW species contained an average of ca. 490 B atoms/mAb molecule, as determined by amino acid analysis. Biodistribution data were collected using nude mice bearing LS174T xenografts and 125I-labeled mAb conjugates. While the lightly B-loaded dipeptide conjugate gave biodistribution results which resembled those of native T84.66 mAb, the undecapeptide conjugate displayed greatly enhanced liver uptake and decreased tumor accretion. These results suggest that as the boron-containing burden on the supporting immunoprotein is greatly increased, as in the case of the T84.66-2 conjugate, loss of circulating conjugate to liver effectively competes with the desired tumor localization. Means which might be taken to circumvent this difficulty have been described elsewhere (ref 15).     

Cyclic AMP phosphodiesterase activity in fetal and adult muscle of the rhesus monkey.

Total cyclic AMP phosphodiesterase activity of voluntary skeletal muscle of the rhesus monkey was highest in the 100-day fetal series, decreased near term, and was lowest in the adult series. Kinetic data indicated the existence of two cyclic AMP phosphodiesterase enzymes in both the fetal and adult muscle. The apparent K_m values for the high-affinity phosphodiesterase were similar in the 100-day fetal and adult skeletal muscle, whereas those for the low-affinity enzyme were twofold higher in the fetal series. The V_max of the high K_m enzyme was tenfold higher in the fetal than in the adult series and the low K_mV_max was fourfold higher in the fetal series. Both caffeine and theophylline were competitive inhibitors of the low K_m phosphodiesterase activity and noncompetitive inhibitors of the high K_m enzyme. No difference was observed in the sensitivity of the fetal and adult enzyme preparations to the methylxanthines or to Ro20-1724.     

Regulation of synthesis of ribosomal proteins during pyrimidine starvation in Escherichia coli.

The synthesis of ribosomal proteins during pyrimidine starvation was investigated. A progressive turn-off of protein synthesis, with a decay half-time of about 5 min, was observed when Escherichia coli cells were starved of uridine. By means of double-labelling, the syntheses of different ribosomal proteins were shown to be turned off unequally during the starvation. Comparison of the turn-off patterns for some proteins and the known polycistronic organization of the structural genes for these proteins suggests that a major cause of the unequal turn-off was the degradation of mRNA molecules for the ribosomal proteins from the 5'-end toward the 3'-end.     

Floristic variation and willow carr development within a southwest England wetland

Abstract. Woodland colonization on wetlands is considered to have a detrimental effect on their ecological value, even though detailed analysis of this process is lacking. This paper provides an evaluation of the ecological changes resulting from succession of poor fen (base‐poor mire) to willow wet woodland on Goss Moor NNR in Cornwall, UK. Different ages of willow carr were associated with eight understorey communities. During willow colonization, in the ground flora, there was a progressive decrease in poor fen species and an associated increase in woodland species, which appeared to be related to an increase in canopy cover and therefore shade. The most diverse community was found to be the most recent willow and was dominated by poor fen species. The oldest willow was the second most diverse and was associated with a reduction in poor fen species and an increase in woodland species. Architectural features were used successfully to assess the general condition and structure of willow. Tree height and DBH were identified as useful parameters to accurately assess willow age in the field. The implications of active intervention to remove willow in order to conserve the full range of communities within the hydrosere are discussed.     

Effects of an African grass invasion on Hawaiian shrubland nitrogen biogeochemistry

African perennial C₄ grasses are highly successful invaders in Hawaiian ecosystems. We examined the effects of African molasses grass (Melinis minutiflora Beauv.) on Hawaiian shrubland nitrogen (N) dynamics without the influence of fire disturbance. Vegetation tissue carbon and nitrogen chemistry, soil inorganic N pools, net N mineralization rates, and total soil N were studied in three adjacent areas: a monospecificMelinis grassland, a mixed grass/shrubland mosaic, and an un-invaded shrubland.Melinis plots within the mosaic area exhibited the largest inorganic N pools and fastest net N mineralization rates, but were temporally variable with grass phenology. Un-invaded shrubland plots contained the smallest inorganic N pools and lowest net N mineralization rates. Grass foliar C:N and litter C:N were lower than those of common shrubland species, providing one possible link between species and ecosystem N dynamics at this site. The combined effects of N cycle modification, successful light competition, and fire-cycle enhancement make the invasion ofMelinis a significant perturbation to Hawaiian shrubland ecosystem function and successional dynamics. ei]Section editor H Lambers     

Dopamine D2-Receptor Isoforms Expressed in AtT20 Cells Inhibit Q-Type High-Voltage-Activated Ca2+ Channels via a Membrane-Delimited Pathway

Abstract : Dopamine D₂ receptors both acutely and chronically inhibit high-voltage-activated Ca²⁺ channels (HVA-CCs). Two alternatively spliced isoforms, D_2L (long) and D_2S (short), are expressed at high levels in rat pituitary intermediate lobe melanotropes but are lacking in anterior lobe corticotropes. We stably transfected D_2L and D_2S into corticotrope-derived AtT20 cells. Both isoforms coupled to inhibition of Q-type calcium channels through pertussis toxin-sensitive G proteins. Thus, we have created a model system in which to study the kinetics of D₂-receptor regulation of Ca²⁺ channels. Rapid inhibition of HVA-CCs was characterized using a novel fluorescence video imaging technique for the measurement of millisecond kinetic events. We measured the time elapsed (lag time) between the arrival of depolarizing isotonic 66 m M K⁺, sensed by fluorescence from included carboxy-X-rhodamine (CXR), and the beginning of increased intracellular Ca²⁺ levels (sensed by changes in indo 1 fluorescence ratio). The lag time averaged 350-550 ms, with no significant differences among cell types. Addition of the D₂-agonist quinpirole (250 μ M ) to the K⁺/CXR solution significantly increased the lag times for D₂-expressing cells but did not alter the lag time for AtT20 controls. The increased lag times for D_2L - and D_2S-transfected cells suggest that at least a fraction of the Ca²⁺ channels was inhibited within the initial 350-550 ms. As this inhibition time is too fast for a multistep second messenger pathway, we conclude that inhibition occurs via a membrane-delimited diffusion mechanism.