Expression of Mitochondrial Regulators PGC1α and TFAM as Putative Markers of Subtype and Chemoresistance in Epithelial Ovarian Carcinoma

Epithelial ovarian carcinoma (EOC), the major cause of gynaecological cancer death, is a heterogeneous disease classified into five subtypes. Each subtype has distinct clinical characteristics and is associated with different genetic risk factors and molecular events, but all are treated with surgery and platinum/taxane regimes. Tumour progression and chemoresistance is generally associated with major metabolic alterations, notably altered mitochondrial function(s). Here, we report for the first time that the expression of the mitochondrial regulators PGC1α and TFAM varies between EOC subtypes; furthermore, we have identified a profile in clear-cell carcinoma consisting of undetectability of PGC1α/TFAM, and low ERα/Ki-67. By contrast, high-grade serous carcinomas were characterised by a converse state of PGC1α/TFAM, ERα positivity and a high Ki-67 index. Interestingly, loss of PGC1α/TFAM and ERα was found also in a non-clear cell EOC cell line made highly resistant to platinum in vitro. Similar to clear-cell carcinomas, these resistant cells also showed accumulation of glycogen. Altogether, our data provide mechanistic insights into the chemoresistant nature of ovarian clear-cell carcinomas. Furthermore, these findings corroborate the need to take into account the diversity of EOC and to develop subtype specific treatment strategies.     

Structural and functional characterization of second-coordination sphere mutants of soybean lipoxygenase-1.

Lipoxygenases are an important class of non-heme iron enzymes that catalyze the hydroperoxidation of unsaturated fatty acids. The details of the enzymatic mechanism of lipoxygenases are still not well understood. This study utilizes a combination of kinetic and structural probes to relate the lipoxygenase mechanism of action with structural modifications of the iron's second coordination sphere. The second coordination sphere consists of Gln(495) and Gln(697), which form a hydrogen bond network between the substrate cavity and the first coordination sphere (Asn(694)). In this investigation, we compared the kinetic and structural properties of four mutants (Q495E, Q495A, Q697N, and Q697E) with those of wild-type soybean lipoxygenase-1 and determined that changes in the second coordination sphere affected the enzymatic activity by hydrogen bond rearrangement and substrate positioning through interaction with Gln(495). The nature of the C-H bond cleavage event remained unchanged, which demonstrates that the mutations have not affected the mechanism of hydrogen atom tunneling. The unusual and dramatic inverse solvent isotope effect (SIE) observed for the Q697E mutant indicated that an Fe(III)-OH(-) is the active site base. A new transition state model for hydrogen atom abstraction is proposed.     

Dealing with errors in interactive sequencing by hybridization.

MOTIVATION: A realistic approach to sequencing by hybridization must deal with realistic sequencing errors. The results of such a method can surely be applied to similar sequencing tasks. RESULTS: We provide the first algorithms for interactive sequencing by hybridization which are robust in the presence of hybridization errors. Under a strong error model allowing both positive and negative hybridization errors without repeated queries, we demonstrate accurate and efficient reconstruction with error rates up to 7%. Under the weaker traditional error model of Shamir and Tsur (Proceedings of the Fifth International Conference on Computational Molecular Biology (RECOMB-01), pp 269-277, 2000), we obtain accurate reconstructions with up to 20% false negative hybridization errors. Finally, we establish theoretical bounds on the performance of the sequential probing algorithm of Skiena and Sundaram (J. Comput. Biol., 2, 333-353, 1995) under the strong error model. AVAILABILTY: Freely available upon request. CONTACT: skiena@cs.sunysb.edu.     

Antilipolytic activity of viprostol, a transdermally active antihypertensive PGE2 analog

Viprostol, a novel prostaglandin E2 congener, was assessed for in vitro antilipolytic activity in the spontaneously obese rat. In isolated epididymal adipocytes, viprostol exhibited a dose-dependent inhibition of catecholamine-stimulated lipolysis at concentrations ranging from 10 microM to 1 mM, but was ineffective at lower concentrations. Additionally, viprostol exhibited approximately 50% of the antilipolytic activity of naturally-occurring PGE1 and PGE2 at similar concentrations, but was as potent as PGF2 alpha. At 10 microM, viprostol inhibited maximum catecholamine-stimulated lipolysis by approximately 35% of the total, hormone-stimulated glycerol release. The results of these experiments indicate that viprostol exhibits antilipolytic activity in vitro, but is less potent than the naturally-occurring PGE's to which it is most closely related structurally.     

Drought Stress and Elevated CO(2) Effects on Soybean Ribulose Bisphosphate Carboxylase Activity and Canopy Photosynthetic Rates

Soybean (Glycine max [L.] cv Bragg) was grown at 330 or 660 microliters CO₂ per liter in outdoor, controlled-environment chambers. When the plants were 50 days old, drought stress was imposed by gradually reducing irrigation each evening so that plants wilted earlier each succeeding day. On the ninth day, as the pots ran out of water CO₂ exchange rate (CER) decreased rapidly to near zero for the remainder of the day. Both CO₂-enrichment and drought stress reduced the total (HCO₃⁻/Mg²⁺-activated) extractable ribulose-1,5-bisphosphate carboxylase (RuBPCase) activity, as expressed on a chlorophyll basis. In addition, drought stress when canopy CER values and leaf water potentials were lowest, reduced the initial (nonactivated) RuBPCase activity by 50% compared to the corresponding unstressed treatments. This suggests that moderate to severe drought stress reduces the in vivo activation state of RuBPCase, as well as lowers the total activity. It is hypothesized that stromal acidification under drought stress causes the lowered initial RuBPCase activities. The K_m(CO₂) values of activated RuBPCase from stressed and unstressed plants were similar; 15.0 and 12.6 micromolar, respectively. RuBP levels were 10 to 30% lower in drought stressed as compared to unstressed treatments. However, RuBP levels increased from near zero at night to around 150 to 200 nanomoles per milligram chlorophyll during the day, even as water potentials and canopy CERs decreased. This suggests that the rapid decline in canopy CER cannot be attributed to drought stress induced limitations in the RuBP regeneration capability. Thus, in soybean leaves, a nonstomatal limitation of leaf photosynthesis under drought stress conditions appears due, in part, to a reduction of the in vivo activity of RuBPCase. Because initial RuBPCase activities were not reduced as much as canopy CER values, this enzymic effect does not explain entirely the response of soybean photosynthesis to drought stress.     

l-Phenylalanyl-l-Glutamate-Stimulated, Chloride-Dependent Glutamate Binding Represents Glutamate Sequestration Mediated by an Exchange System

Stimulation of glutamate binding by the dipeptide L-phenylalanyl-L-glutamate (Phe-Glu) was inhibited by the peptidase inhibitor bestatin, suggesting that the stimulation was caused by glutamate liberated from the dipeptide and not by the dipeptide itself. It further suggests that this form of glutamate binding should be reinterpreted as glutamate sequestration and that stimulation of binding both by dipeptides and after preincubation with high concentrations of glutamate is likely to be due to counterflow accumulation. Several other criteria indicate that most of glutamate binding stimulated by chloride represents glutamate sequestration: Binding is reduced when the osmolarity of the incubation medium is increased, when membranes incubated with [3H]glutamate are lysed before filtration, and when membranes are made permeable by transient exposure to saponin. Moreover, dissociation of bound glutamate after a 100-fold dilution of the incubation medium is accelerated about 50 times by the addition of glutamate to the dilution medium. This result would be anomalous if glutamate were bound to a receptor site; it suggests instead that glutamate is transported in and out of membrane vesicles by a transport system that preferentially mediates exchange between internal and external glutamate. Glutamate binding contains a component of glutamate sequestration even when measured in the absence of chloride. Sequestration is adequately abolished only after treating membranes with detergents; even extensive lysis, sonication, and freezing/thawing may be insufficient.     

Immunochemical characterization of two antigenic sites on human apolipoprotein A-I; localization and lipid modulation of these epitopes

Two monoclonal antibodies, A17 and A30, were raised against human apolipoprotein A-I (apo A-I). They were studied by competitive inhibition of 125I-labeled HDL3 with HDL subfractions, delipidated apo A-I, and complexes of dimyristoylphosphatidylcholine (DMPC) containing apo A-I and apo A-II. Immunoblotting located the A17 antibody on CNBr fragment 4 of apo A-I and the A30 antibody on CNBr fragment 1. The A17 antigenic determinant was expressed identically in all HDL subclasses, on delipidated apo A-I as well as all on the DMPC-apo A-I and DMPC-apo A-I/apo A-II complexes. In contrast, the apparent affinity constant of the A30 antibody for delipidated apo A-I was about 30-times less than for HDL3 or for apo A-I/apo A-II-phospholipid complexes. These data suggest that the association of apo A-I with phospholipids improves the reactivity of the A30 monoclonal antibody towards apo A-I, and that this antigenic determinant has a different conformation in delipidated apo A-I compared to apo A-I complexed with phospholipids. Turbidimetric and fluorescence experiments monitoring the phospholipid-apo A-I association in the presence and in the absence of the A17 and A30 antibodies were consistent with the competition experiments carried out by solid phase radioimmunoassay (RIA). After reaction of apo A-I with the A30 antibody, we observed an enhancement of the degradation kinetics of large multilamellar vesicles (LMV), while the A17 antibody did not have a significant effect. Calcein leakage experiments carried out below the transition temperature of DPPC showed an enhancement of the degradation kinetics with both monoclonal antibodies, while the phase-transition release was independent of the reaction of apo A-I with the monoclonal antibodies. These data therefore suggest the existence of at least two different types of epitope on apo A-I, which might account for the differences in immunological reactivity of apo A-I that is either delipidated or present on HDL.     

Photosynthetic Activity in the Flower Buds of ;Valencia' Orange (Citrus sinensis [L.] Osbeck)

Flower buds of `Valencia' orange (Citrus sinensis [L.] Osbeck) were able to fix ¹⁴CO₂ into a number of compounds in their own tissues under both light and dark conditions. The total incorporation, however, was about 4-fold higher in the light than in the dark. In the light, 50% of the total ¹⁴C label was found in the neutral fraction (sugars), 22% in the basic fraction (amino acids), and 26% in the acid-1 fraction (organic acids). In the dark, about 95% of the ¹⁴C label was incorporated into the basic and acid-1 fractions. Activities of ribulose bisphosphate carboxylase and phosphoenolpyruvate carboxylase (expressed in micromoles CO₂ per milligram protein per hour) averaged 1.95 and 8.87 for the flower buds, and 28.5 and 3.6 for the leaves, respectively. The ability of orange flower buds to fix ambient CO₂ into different compounds suggests that this CO₂ assimilation may have some regulatory role during the early reproductive stages in determining citrus fruit initiation and setting.