Inhibition of prolyl hydroxylase by poly(ADP-ribose) and phosphoribosyl-AMP. Possible role of ADP-ribosylation in intracellular prolyl hydroxylase regulation

Poly(ADP-ribose) prepared by incubating NAD+ with rat liver nuclei inhibited the hydroxylation reaction catalyzed by purified prolyl hydroxylase (proline,2-oxoglutarate dioxygenase, EC 1.14.11.2) in vitro. Near complete inhibition of the enzyme was seen in the presence of 6 nM (ADP-Rib)18 with a Ki(app) of 1.5 nM. The monomer unit of poly(ADP-ribose), adenosine diphosphoribose (ADP-Rib), was found to be a weak inhibitor. On the other hand, poly(ADP-ribose)-derived phosphoribosyl-AMP (PRib-AMP) and its dephosphorylated product, ribosyl-ribosyl-adenine (Rib-RibA), inhibited the enzyme in nanomolar concentrations (Ki(app) 16.25 nM). The order of inhibition was (ADP-Rib)18 greater than PRib-AMP, Rib-RibA much greater than ADP-Rib. These results suggested that the 1"----2' ribosyl-ribosyl moiety in these compounds was involved in the inhibition of the enzyme. The possibility that intracellular prolyl hydroxylase is regulated by the involvement of ADP-ribosylation reactions was examined in confluent cultures of skin fibroblast treated with 20 mM lactate. The activity of prolyl hydroxylase was stimulated by 145% over that of untreated cultures. In the lactate-treated cells, the level of NAD+ was lowered and the total ADP-ribosylation of cellular proteins reduced by 40%. These observations imply that the lactate-induced activation of cellular prolyl hydroxylase is mediated by a reduction in ADP-ribosylation and that the synthesis and degradation of ADP-ribose moiety(ies) may possibly regulate prolyl hydroxylase activity in vivo.     

Re-evalution of “Typothorax”meadei,a Late Triassic aetosaur from the United States

Aetosaur specimens from Howard County, Texas, USA, namedTypothorax meadei by Sawin (1947) represent a new genus here namedLongosuchus (type and only species -L. meadei). Longosu-chus is characterised by the possession of seven dentary teeth, a dentary excluded from the ventral margin of the lateral mandibular fenestra and by the angular, spinose lateral scutes throughout the dorsal region with lateral horns which have faceted sides.Longosuchus is found in mid-late Carnian strata in Texas, New Mexico and North Carolina, USA. Aetosaurs can be used to distinguish three successive biochrons in Late Triassic strata of the American Southwest, which are, in ascending order of age, theLongosuchus biochron, theStagonolepis biochron and theTypothorax biochron.     

Reduction of 3-methoxytyramine concentrations in the caudate nucleus of rats after exposure to high-energy iron particles: evidence for deficits in dopaminergic neurons

Exposure to low doses of high-energy iron particles can alter motor behavior. The ability of rats to hang from a wire has been reported to be significantly degraded after exposure to doses as low as 0.5 Gy. In addition, deficits in the ability of acetylcholine to regulate dopamine release in the caudate nucleus (an area in the brain important for motor function) have been found. The concentrations of 3-methoxytyramine (3-MT), a metabolite of dopamine whose concentrations reflect dopamine release in vivo, were measured after rats were exposed to different doses of high-energy iron particles to gain further information about the effect of radiation on the dopaminergic system. Concentrations of 3-MT were significantly reduced 3 days after exposure to 5 Gy but returned to control values by 8 days. After 6 months, concentrations were again less than control values. Exposure to 5 Gy of high-energy electrons or gamma photons had no effect 3 days after exposure. Very high doses of electrons were needed to alter 3-MT concentrations. One hundred grays of electrons decreased 3-MT 30 min after irradiation but levels returned to control values by 60 min. Gamma photons had no effect after doses up to 200 Gy. These results provide further evidence that exposure to heavy particles can degrade motor behavior through an action on dopaminergic mechanisms and that this can occur after doses much lower than those needed for low-LET radiation.     

Involvement of prostaglandins and histamine in radiation-induced temperature responses in rats

Exposure of rats to 1-15 Gy of gamma radiation induced hyperthermia, whereas exposure to 20-150 Gy produced hypothermia. Since radiation exposure induced the release of prostaglandins (PGs) and histamine, the role of PGs and histamine in radiation-induced temperature changes was examined. Radiation-induced hyper- and hypothermia were antagonized by pretreatment with indomethacin, a cyclooxygenase inhibitor. Intracerebroventricular administration of PGE2 and PGD2 induced hyper- and hypothermia, respectively. Administration of SC-19220, a specific PGE2 antagonist, attenuated PGE2- and radiation-induced hyperthermia, but it did not antagonize PGD2- or radiation-induced hypothermia. Consistent with an apparent role of histamine in hypothermia, administration of disodium cromoglycate (a mast cell stabilizer), mepyramine (H1-receptor antagonist), or cimetidine (H2-receptor antagonist) attenuated PGD2- and radiation-induced hypothermia. These results suggest that radiation-induced hyperthermia is mediated via PGE2 and that radiation-induced hypothermia is mediated by another PG, possibly PGD2, via histamine.     

Effect of vascular cannulation on serum concentrations of LH, FSH, prolactin and cortisol in prepubertal gilts

Two experiments were conducted to determine whether cannulation of the jugular vein in gilts alters serum concentrations of LH, FSH, prolactin (PRL) or cortisol (C). In Experiment 1, 12 crossbred prepubertal gilts weighing 95 +/- 1.3 kg were immobilized by snaring, and tygon tubing was threaded into the anterior vena cava through a 12-gauge needle inserted into the jugular vein. Five hours later, blood samples were drawn at 20-min intervals for 4 h (Day 0). Samples were also drawn at 20-min intervals for 4-h periods 24 h (Day 1) and 48 h (Day 2) after cannulation. Serum concentrations of LH were similar (P=0.26) among Day 0 (0.40 ng/ml), Day 1 (0.39 ng/ml) and Day 2 (0.34 ng/ml). Serum PRL was similar (P=0.07) among Day 0 (4.10 ng/ml), Day 1 (3.87 ng/ml) and Day 2 (3.43 ng/ml). Serum concentrations of C were greater (P < 0.001) on Day 0 (8.32 ng/ml) than Day 1 (4.48 ng/ml) or Day 2 (3.54 ng/ml). In Experiment 2, cannulas were placed in 29 prepubertal gilts. Two days after initial cannulation, six blood samples were drawn at 20-min intervals. Gilts were then immobilized by snaring, and a second cannulae was inserted into the contralateral vein. Five blood samples were taken at 2-min intervals during the second cannulation and then six samples were drawn at 20-min intervals. Serum LH and FSH were not altered by cannulation or elevated during the subsequent 2-h sampling period (P>0.05). In contrast, serum concentrations of PRL rose slowly (P<0.05) during cannulation and remained elevated for 60 min before returning to baseline. Serum concentrations of C rose within 6 min of cannulation, remained elevated for 30 min, and then declined over the next 90 min. From these two experiments, it appears that secretory patterns of LH and FSH can be accurately assessed immediately after cannulation in prepubertal gilts. Measurements of serum PRL and C that reflect nonstressed conditions, however, cannot be obtained until at least 2 h or 1 d after cannulation, respectively.     

Molten globule monomer to condensed dimer: role of disulfide bonds in platelet factor-4 folding and subunit association.

Platelet factor 4 (PF4) exhibits high affinity for heparin and exists as a tetramer in solution under physiologic conditions. Reduction of the two disulfide bridges in PF4 increases the protein's dissociation constant for heparin approximately 20-fold and shifts the highest apparent aggregation state from tetramer to dimer as evidenced by gel filtration, chemical cross-linking, and 1H-NMR studies. 1H-NMR spectra of reduced PF4 monomers generally show narrower, less dispersed, upfield-shifted NH and alpha H resonances, suggesting the presence of an unfolded monomer state. Reduced PF4 monomer folding, however, is evidenced by the presence of about 12 relatively long-lived backbone NHs and by CD spectra that indicate conservation of overall secondary structure. These data suggest the presence of a molten globule-type state. Urea denaturation shifts this apparent molten globule to a fully unfolded state characterized by more random coil-like resonance shifts. The reduced PF4 dimer state yields NMR and CD data consistent with preservation of tertiary structural folds found for the native species. In this regard, the reduced PF4 folding transition is thermodynamically linked with dimer formation which stabilizes tertiary structure. Monomer-dimer association equilibria for reduced PF4 essentially follow the same pH and salt titration trends as reported previously for native PF4 dimers [Mayo, K. H., & Chen, M. J. (1989) Biochemistry 28, 9469-9478], indicating that that dimer interface is generally conserved in the absence of disulfide constraints. Reduced PF4 tetramers are not apparent under any conditions investigated, suggesting that disulfides are necessary for efficient antiparallel beta-sheet alignment between dimer pairs.     

Activation of recA protein: The salt-induced structural transition

Purified recA protein is induced by high salt concentrations to hydrolyse ATP even in the absence of DNA. By small angle neutron scattering we show that this salt activation results from a structural transition of the protein filament in the presence of ATPγS from the inactive, compact form (a helical polymer of pitch 70 Å and cross-sectional radius of gyration R_c 40 Å) to the open form (a helical filament of pitch 95 Å and R_c 35 Å, which are the same structural parameters as in the ATPase active complex with DNA and ATP), without detectable change in the degree of association. We conclude that activation of recA is due to the same structural change whether induced by the binding of DNA or by salt. Indeed, the other enzymatic activity of recA, the proteolytic cleavage of the lexA repressor, is found to be inducible by the same salt concentrations as those of the structural transition.     

The 'destruction box' of cyclin A allows B-type cyclins to be ubiquitinated, but not efficiently destroyed.

The destruction of mitotic cyclins by programmed proteolysis at the end of mitosis is an important element in cell cycle control. This proteolysis depends on a conserved motif of nine residues known as the 'destruction box', which is located 40-50 residues from the N-terminus. The sequences of the A- and B-type destruction boxes are slightly different, which might account for the differences in timing of their destruction. When the cyclin A-type destruction box was substituted for the normal one in cyclin B1 or B2, however, the resulting constructs were unexpectedly stable, although the converse substitution of B-type destruction boxes in cyclin A permitted normal degradation. We compared the ubiquitination of various cyclin constructs, and found that whereas mutation of the highly conserved residues in the destruction box strongly reduced the level of ubiquitinated intermediates, the stable destruction box 'swap' constructs did form such adducts. Thus, while ubiquitination is probably necessary for cyclin destruction, it is not sufficient. We also found that poly-ubiquitinated cyclin derivatives are still bound to p34cdc2, which is not detectably ubiquitinated itself, raising the questions of how cyclin and cdc2 dissociate from one another, and at what stage, in the process of degradation.     

The role of proteolysis in cell cycle progression in Schizosaccharomyces pombe.

A cell-free system derived from Xenopus eggs was used to identify the 'destruction box' of the Schizosaccharomyces pombe B-type cyclin, Cdc13, as residues 59-67: RHALDDVSN. Expression of indestructible Cdc13 from a regulated promoter in S.pombe blocked cells in anaphase and inhibited septation, showing that destruction of Cdc13 is necessary for exit from mitosis, but not for sister chromatid separation. In contrast, strong expression of a polypeptide comprising the N-terminal 70 residues of Cdc13, which acts as a competitive inhibitor of destruction box-mediated proteolysis, inhibited both sister chromatid separation and the destruction of Cdc13, whereas an equivalent construct with a mutated destruction box did not. Appropriately timed expression of this N-terminal fragment of Cdc13 overcame the G1 arrest seen in cdc10 mutant strains, suggesting that proteins required for the initiation of S phase are subject to destruction by the same proteolytic machinery as cyclin.     

Genetically stable cell lines of cucumber for the large-scale production of diploid somatic embryos

For the initiation of embryogenic cucumber ( Cucumis sativus L.) cell lines, from excised radicles, directly in liquid medium, the culture regime, explant density and type and concentration of hormones were adjusted so that pro-embryogenic masses (PEMs) were formed within about 8 weeks. The established cucumber cell lines were maintained tor several years without loss of embryogenic and genetic stability. The ploidy level of somatic embryos from different cucumber eell lines was either diploid or tetraploid and depended on the ploidy level of Ihe cell line. Cucumber cell lines that produced only diploid embryos were obtained by selecting completely diploid explant material and growing it in the dark during the initiation phase. Mixoploid explains could lead to tetraploid or mixoploid ceil lines. Isolation and additional selection and subculturing of single PEMs resulted in either completely diploid or tetraploid cell lines, indicating that all cells of individual PEMs are either diploid or tetraploid. The ernbryogenic cucumber cell Imes, differing only in ploidy level, were indistinguishable in growth rate and embryogetiic potential and were genetically stable over several years.