EFFECTS OF SUCROSE ON METABOLIZE POOL SIZES AND RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE ACTIVITY IN STRAWBERRY PLANTLETS CULTIVATED IN VITRO

To identify the physiological and biochemical events leading to the negative effects of sucrose in culture medium on the photosynthetic capacity of plantlets cultivated in vitro, time-course changes in photosynthesis, metabolize pool sizes, and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity were investigated in strawberry (Fragaria × ananassa Duch. cv. Kent) plantlets following their transfer to medium with or without sucrose. When the plantlets grown in medium without sucrose were transferred to a similar medium with 30 g sucrose/liter, their net photosynthesis decreased and their level of phosphorylated compounds increased with time. In addition, initial Kcat, total Kcat, and the activation state of Rubisco decreased in these plantlets. Conversely, when the plantlets grown in medium with 30 g sucrose/liter were transferred to a similar medium without sucrose, their net photosynthesis slowly increased with time and their level of phosphorylated compounds slowly decreased. A slow increase with time of initial Kcat, total Kcat, and the activation state of Rubisco was also observed in these plantlets. The results of the present research suggest that the reduced photosynthetic capacity of strawberry plantlets cultivated in vitro in the presence of sucrose was the consequence of reduced Rubisco efficiency due to its deactivation and the possible presence of a putative tight binding inhibitor.

Download Full-text

Photometric Measurements of Rubisco Activity in Leaves of Deciduous Fruit Crops

HortScience ◽

10.21273/hortsci.32.3.531a ◽

1997 ◽

Vol 32 (3) ◽

pp. 531A-531

Author(s):

Lailiang Cheng ◽

Leslie H. Fuchigami

Keyword(s):

Net Photosynthesis ◽

Photometric Method ◽

Ribulose Bisphosphate Carboxylase ◽

Fruit Crops ◽

Rubisco Activity ◽

Bisphosphate Carboxylase ◽

Ribulose Bisphosphate Carboxylase Oxygenase ◽

Deciduous Fruit ◽

Photometric Measurements

Ribulose bisphosphate carboxylase/oxygenase (Rubisco) initiates the photosynthetic carbon metabolism;therefore, its activity has been measured in many physiological studies. However, information on in vitro Rubisco activity from leaves of deciduous fruit crops is very limited and the reported activities are suspiciously low. We measured Rubisco activity in crude extracts of leaves of apple, pear, peach, cherry, and grape by using a photometric method in which RuBP carboxylation was enzymically coupled to NADH oxidation. Replacing polyvinylpyrrolidone with polyvinylpolypyrrolidone in the extraction solution significantly increased extractable Rubisco activity. Depending on species, freezing leaf discs in liquid nitrogen followed by storage at –80°C for only 24 hr reduced both initial and total Rubisco activity to 5% to 50% of that obtained from fresh leaves. Initial Rubisco activity from fresh leaf tissues of all species was well correlated with maximum Rubisco activity (Vcmax) estimated from gas exchange; an exception was pear, where initial Rubisco activity was higher than Vcmax. In most cases, initial Rubisco activity was approximately two to three times higher than net photosynthesis.

Download Full-text

Differences in isoprenoid-mediated energy dissipation pathways between coastal and interior Douglas-fir seedlings in response to drought

Tree Physiology ◽

10.1093/treephys/tpz075 ◽

2019 ◽

Vol 39 (10) ◽

pp. 1750-1766 ◽

Cited By ~ 2

Author(s):

Laura Verena Junker-Frohn ◽

Anita Kleiber ◽

Kirstin Jansen ◽

Arthur Gessler ◽

Jürgen Kreuzwieser ◽

...

Keyword(s):

Energy Dissipation ◽

Intraspecific Variation ◽

Photosynthetic Capacity ◽

Photosynthetic Apparatus ◽

Douglas Fir ◽

Future Climate ◽

Photochemical Quenching ◽

Negative Effects ◽

Climate Conditions ◽

Pool Sizes

ABSTRACT Plants have evolved energy dissipation pathways to reduce photooxidative damage under drought when photosynthesis is hampered. Non-volatile and volatile isoprenoids are involved in non-photochemical quenching of excess light energy and scavenging of reactive oxygen species. A better understanding of trees’ ability to cope with and withstand drought stress will contribute to mitigate the negative effects of prolonged drought periods expected under future climate conditions. Therefore we investigated if Douglas-fir (Pseudotsuga menziesii(Mirb.)) provenances from habitats with contrasting water availability reveal intraspecific variation in isoprenoid-mediated energy dissipation pathways. In a controlled drought experiment with 1-year-old seedlings of an interior and a coastal Douglas-fir provenance, we assessed the photosynthetic capacity, pool sizes of non-volatile isoprenoids associated with the photosynthetic apparatus, as well as pool sizes and emission of volatile isoprenoids. We observed variation in the amount and composition of non-volatile and volatile isoprenoids among provenances, which could be linked to variation in photosynthetic capacity under drought. The coastal provenance exhibited an enhanced biosynthesis and emission of volatile isoprenoids, which is likely sustained by generally higher assimilation rates under drought. In contrast, the interior provenance showed an enhanced photoprotection of the photosynthetic apparatus by generally higher amounts of non-volatile isoprenoids and increased amounts of xanthophyll cycle pigments under drought. Our results demonstrate that there is intraspecific variation in isoprenoid-mediated energy dissipation pathways among Douglas-fir provenances, which may be important traits when selecting provenances suitable to grow under future climate conditions.

Download Full-text

Purification and characterization of NAD+:ADP-ribosyltransferase (polymerizing) from Dictyostelium discoideum

Biochemical Journal ◽

10.1042/bj2930275 ◽

1993 ◽

Vol 293 (1) ◽

pp. 275-281 ◽

Cited By ~ 16

Author(s):

B Kofler ◽

E Wallraff ◽

H Herzog ◽

R Schneider ◽

B Auer ◽

...

Keyword(s):

Dictyostelium Discoideum ◽

Time Course ◽

Affinity Purification ◽

Tight Binding ◽

Purification Scheme ◽

Adp Ribosyltransferase ◽

Dna Ends ◽

Lower Eukaryote

A novel affinity-purification scheme based on the tight binding of NAD+:ADP-ribosyltransferase (polymerizing) [pADPRT; poly(ADP-ribose) polymerase; EC 2.4.2.30] to single-strand nicks in DNA, single-stranded patches and DNA ends has been developed to facilitate the purification of this enzyme from the lower eukaryote Dictyostelium discoideum. Two homogeneous forms of the enzyme, with M(r) values of 116,000 and 90,000, were prepared from D. discoideum by using poly(A) hybridized to oligo(dT)-cellulose as affinity material. The Km is 20 microM NAD+ for the 90,000-M(r) protein and 77 microM NAD+ for the 116,000-M(r) protein. The optimum conditions for the enzyme activity in vitro are 6-10 degrees C and pH 8. The time course is linear during the first 10 min of the reaction only. As in enzymes of higher eukaryotes, the activity is dependent on DNA and histone H1 and is inhibited by 3-methoxybenzamide, nicotinamide, theophylline, caffeine and thymidine.

Download Full-text

Iron release from recombinant N-lobe and single point Asp63 mutants of human transferrin by EDTA

Biochemical Journal ◽

10.1042/bj3280439 ◽

1997 ◽

Vol 328 (2) ◽

pp. 439-445 ◽

Cited By ~ 11

Author(s):

Qing-Yu HE ◽

B. Anne MASON ◽

C. Robert WOODWORTH

Keyword(s):

Single Point ◽

Tight Binding ◽

Structural Factors ◽

Iron Release ◽

Negative Effects ◽

Major Pathway ◽

Negative Effect ◽

Decreasing Ph ◽

Kinetic Mode

Transferrins bind ferric ion and deliver the iron to cells. The mechanism of the iron release has been studied kinetically, in vitro, with the aid of single point mutants in which the iron-binding ligand, Asp63 (aspartic acid-63, D63), has been changed to Ser, Asn, Glu and Ala. Iron release from the unmutated N-lobe of human serum transferrin (hTF/2N) by EDTA is influenced by a variety of factors. The rate-determining conformational-change mechanism may be a major pathway for iron release from hTF/2N's having a ‘closed’ conformation, which leads to a saturation kinetic mode with respect to ligand concentration. The effect of chloride depends on the protein conformation, showing a negative action in the case of tight binding and a positive action when the protein has an ‘open’ or ‘loose’ conformation. The negative effect of chloride could originate from the binding competition between chloride and the chelate to the active site for iron release, and the positive effect could derive from the synergistic participation of chloride in iron removal. The ‘open’ conformation may be induced by decreasing pH: the transitional point appears to be at about pH 6.3 for the wild-type hTF/2N; the ‘loose’ conformation may be facilitated by mutations at D63, which result in the loss of a key linking component in interdomain interactions of the protein. In the latter case, structural factors dominate over other potential negative effects because the weak interdomain contacts derived from the mutation of D63 cause the binding site to open easily, even at pH 7.4. Therefore chloride exhibits an accelerating action on iron release by EDTA from all the D63 mutants.

Download Full-text

Changes in ribulose-1,5-bisphosphate carboxylase/oxygenase and phosphoenolpyruvate carboxylase activities and 14CO2 fixation during the rooting of strawberry shoots in vitro

Canadian Journal of Plant Science ◽

10.4141/cjps94-150 ◽

1994 ◽

Vol 74 (4) ◽

pp. 827-831 ◽

Cited By ~ 10

Author(s):

Chafik Hdider ◽

Yves Desjardins

Keyword(s):

Phosphoenolpyruvate Carboxylase ◽

Time Course ◽

Carbon Fixation ◽

Acid Synthesis ◽

Rubisco Activity ◽

Amino Acid Synthesis ◽

Bisphosphate Carboxylase ◽

Rooting Medium ◽

Rapid Incorporation

The potential for carbon fixation was investigated in in vitro strawberry (Fragaria × ananassa Duch. Kent) shoots 5, 10 and 28 d after transfer to a rooting medium. The activities of ribulose-1,5-bisphosphate carboxylase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC) and the time course of 14CO2 fixation were investigated. Five days after transfer, Rubisco activity was low but was increased two-fold after 28 d. In contrast PEPC activity was highest at 5 d and declined to about 0.4-fold by day 28. The rate of 14CO2 fixation was similar at 5, 10 and 28 d after transfer. However, a more rapid incorporation of 14CO2 into amino acids was observed at 5 than at 10 or 28 d after transfer. These results suggest that strawberry shoots undergo a progressive transition from heterotrophic to autotrophic carbon fixation during their rooting and that PEPC plays an important role in sustaining carbon fixation and amino acid synthesis during the first few days after their transfer to rooting medium. Key words: In vitro culture, phosphoenolpyruvate carboxylase, ribulose-1,5-bisphosphate carboxylase

Download Full-text

Comparison of High Purity Factor IX Concentrates and a Prothrombin Complex Concentrate in a Canine Model of Thrombogenicity

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646468 ◽

1991 ◽

Vol 66 (05) ◽

pp. 609-613 ◽

Cited By ~ 14

Author(s):

I R MacGregor ◽

J M Ferguson ◽

L F McLaughlin ◽

T Burnouf ◽

C V Prowse

Keyword(s):

High Purity ◽

Time Course ◽

Prothrombin Complex Concentrate ◽

Degradation Products ◽

Canine Model ◽

Factor Ix ◽

In Vitro Tests ◽

Albumin Infusion

SummaryA non-stasis canine model of thrombogenicity has been used to evaluate batches of high purity factor IX concentrates from 4 manufacturers and a conventional prothrombin complex concentrate (PCC). Platelets, activated partial thromboplastin time (APTT), fibrinogen, fibrin(ogen) degradation products and fibrinopeptide A (FPA) were monitored before and after infusion of concentrate. Changes in FPA were found to be the most sensitive and reproducible indicator of thrombogenicity after infusion of batches of the PCC at doses of between 60 and 180 IU/kg, with a dose related delayed increase in FPA occurring. Total FPA generated after 100-120 IU/kg of 3 batches of PCC over the 3 h time course was 9-12 times that generated after albumin infusion. In contrast the amounts of FPA generated after 200 IU/kg of the 4 high purity factor IX products were in all cases similar to albumin infusion. It was noted that some batches of high purity concentrates had short NAPTTs indicating that current in vitro tests for potential thrombogenicity may be misleading in predicting the effects of these concentrates in vivo.

Download Full-text

The Effect of Heparin vs. Citrate on the Interaction of Platelets with Vascular Graft Materials

Thrombosis and Haemostasis ◽

10.1055/s-0038-1660145 ◽

1985 ◽

Vol 54 (04) ◽

pp. 842-848 ◽

Cited By ~ 14

Author(s):

Kandice Kottke-Marchant ◽

James M Anderson ◽

Albert Rabinovitch ◽

Richard A Huskey ◽

Roger Herzig

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Vascular Graft ◽

Time Course ◽

Platelet Reactivity ◽

Polymer Surfaces ◽

In Vitro Perfusion ◽

Citrate Anticoagulation ◽

Platelet Release

SummaryHeparin is known to affect platelet function in vitro, but little is known about the effect of heparin on the interaction of platelets with polymer surfaces in general, and vascular graft materials in particular. For this reason, the effect of heparin vs. citrate anticoagulation on the interaction of platelets with the vascular graft materials expanded polytetrafluoroethylene (ePTFE), Dacron Bionit (DB) and preclotted Dacron Bionit (DB/PC) was studied in a recirculating, in vitro perfusion system. Platelet activation, as shown by a decrease in platelet count, an increase in platelet release and a decrease in platelet aggregation, was observed for all vascular graft materials tested using heparin and was greater for Dacron and preclotted Dacron than for ePTFE. Significant differences between heparin and citrate anticoagulation were seen for platelet release, platelet aggregation and the relative ranking of material platelet-reactivity. However, the trends and time course of platelet activation were similar with both heparin and citrate for the materials tested.

Download Full-text

Diabetes and Heart Failure: Is it Hyperglycemia or Hyperinsulinemia?

Current Vascular Pharmacology ◽

10.2174/1570161117666190408164326 ◽

2020 ◽

Vol 18 (2) ◽

pp. 148-157 ◽

Cited By ~ 3

Author(s):

Triantafyllos Didangelos ◽

Konstantinos Kantartzis

Keyword(s):

Heart Failure ◽

Insulin Treatment ◽

Close Association ◽

Adverse Outcomes ◽

Negative Effects ◽

Beneficial Effects ◽

Appropriate Treatment ◽

Increased Risk ◽

Treatment Of Diabetes

The cardiac effects of exogenously administered insulin for the treatment of diabetes (DM) have recently attracted much attention. In particular, it has been questioned whether insulin is the appropriate treatment for patients with type 2 diabetes mellitus and heart failure. While several old and some new studies suggested that insulin treatment has beneficial effects on the heart, recent observational studies indicate associations of insulin treatment with an increased risk of developing or worsening of pre-existing heart failure and higher mortality rates. However, there is actually little evidence that the associations of insulin administration with any adverse outcomes are causal. On the other hand, insulin clearly causes weight gain and may also cause serious episodes of hypoglycemia. Moreover, excess of insulin (hyperinsulinemia), as often seen with the use of injected insulin, seems to predispose to inflammation, hypertension, dyslipidemia, atherosclerosis, heart failure, and arrhythmias. Nevertheless, it should be stressed that most of the data concerning the effects of insulin on cardiac function derive from in vitro studies with isolated animal hearts. Therefore, the relevance of the findings of such studies for humans should be considered with caution. In the present review, we summarize the existing data about the potential positive and negative effects of insulin on the heart and attempt to answer the question whether any adverse effects of insulin or the consequences of hyperglycemia are more important and may provide a better explanation of the close association of DM with heart failure.

Download Full-text

Studies on the mechanism of acute inhibition of thyroglobulin hydrolysis by iodine

Acta Endocrinologica ◽

10.1530/acta.0.1080511 ◽

1985 ◽

Vol 108 (4) ◽

pp. 511-517 ◽

Cited By ~ 12

Author(s):

Nandalal Bagchi ◽

Birdie Shivers ◽

Thomas R. Brown

Keyword(s):

Time Course ◽

Period 2 ◽

Iodotyrosine Deiodinase ◽

Rate Of Hydrolysis ◽

Underlying Mechanisms ◽

Excess Iodide ◽

Sites Of Action ◽

Hydrolysis Of

Abstract. Iodine in excess is known to acutely inhibit thyroidal secretion. In the present study we have characterized the time course of the iodine effect in vitro and investigated the underlying mechanisms. Labelled thyroid glands were cultured in vitro in medium containing mononitrotyrosine, an inhibitor of iodotyrosine deiodinase. The rate of hydrolysis of labelled thyroglobulin was measured as the proportion of labelled iodotyrosines and iodothyronines recovered at the end of culture and was used as an index of thyroidal secretion. Thyrotrophin (TSH) administered in vivo acutely stimulated the rate of thyroglobulin hydrolysis. Addition of Nal to the culture medium acutely inhibited both basal and TSH-stimulated thyroglobulin hydrolysis. The effect of iodide was demonstrable after 2 h, maximal after 6 h and was not reversible upon removal of iodide. Iodide abolished the dibutyryl cAMP induced stimulation of thyroglobulin hydrolysis. Iodide required organic binding of iodine for its effect but new protein or RNA synthesis was not necessary. The inhibitory effects of iodide and lysosomotrophic agents such as NH4C1 and chloroquin on thyroglobulin hydrolysis were additive suggesting different sites of action. Iodide added in vitro altered the distribution of label in prelabelled thyroglobulin in a way that suggested increased coupling in the thyroglobulin molecule. These data indicate that 1) the iodide effect occurs progressively over a 6 h period, 2) continued presence of iodide is not necessary once the inhibition is established, 3) iodide exerts its action primarily at a post cAMP, prelysosomal site and 4) the effect requires organic binding of iodine, but not new RNA or protein synthesis. Our data are consistent with the hypothesis that excess iodide acutely inhibits thyroglobulin hydrolysis by increasing the resistance of thyroglobulin to proteolytic degradation through increased iodination and coupling.

Download Full-text

DDRE-09. DEVELOPING IDO-PROTACS TO IMPROVE IMMUNOTHERAPEUTIC EFFICACY IN PATIENTS WITH GLIOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa215.254 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii63-ii63

Author(s):

Lakshmi Bollu ◽

Derek Wainwright ◽

Lijie Zhai ◽

Erik Ladomersky ◽

Kristen Lauing ◽

...

Keyword(s):

Protein Degradation ◽

Time Course ◽

Immune Cell ◽

Enzyme Inhibitors ◽

Tumor Development ◽

Ubiquitin Proteasome System ◽

Degradation Pathway ◽

Specific Protein ◽

Cell Functions

Abstract INTRODUCTION Indoleamine 2,3-dioxygenase 1 (IDO; IDO1) is a rate-limiting enzyme that metabolizes the essential amino acid tryptophan into kynurenine. Recent work by our group has revealed that IDO promotes tumor development and suppresses immune cell functions independent of its enzyme activity. Moreover, pharmacologic IDO enzyme inhibitors that currently serve as the only class of drugs available for targeting immunosuppressive IDO activity, fail to improve the survival of patients with GBM. Here, we developed IDO-Proteolysis Targeting Chimeras (IDO-PROTACs). PROTACs bind to a specific protein and recruit an E3 ubiquitin ligase that enhance proteasome-mediated degradation of the target protein. METHODS A library of ≥100 IDO-PROTACs were developed by joining BMS986205 (IDO binder) with a linker group to various E3-ligase ligands. Western blot analysis of PROTAC-induced IDO degradation was tested in vitro among multiple human and mouse GBM cell lines including U87, GBM6, GBM43 and GL261 along a time course ranging between 1–96 hours of treatment and at varying concentrations. The mechanism of IDO protein degradation was investigated using pharmacologic ligands that inhibit or compete with the proteasome-mediated protein degradation pathway. RESULTS Primary screening identified several IDO-PROTACs with IDO protein degradation potential. Secondary screening showed that our lead compound has a DC50 value of ~0.5µM with an ability to degrade IDO in all GBM cells analyzed, and an initial activity within 12 hours of treatment that extended for up to 96 hours. Mutating the CRBN-binding ligand, pretreatment with the ubiquitin proteasome system inhibitors MG132 or MLN4924 or using unmodified parental compound all inhibited IDO protein degradation. CONCLUSIONS This study developed an initial IDO-PROTAC technology that upon further optimization, can neutralize both IDO enzyme and non-enzyme immunosuppressive effects. When combined with other forms of immunotherapy, IDO-PROTACs have the potential to substantially enhance immunotherapeutic efficacy in patients with GBM.

Download Full-text