Adiponectin multimerization is dependent on conserved lysines in the collagenous domain: Evidence for regulation of multimerization by alterations in posttranslational modifications

Adiponectin is a secreted, multimeric protein with insulin-sensitizing, antiatherogenic, and antiinflammatory properties. Serum adiponectin consists of trimer, hexamer, and larger high-molecular-weight (HMW) multimers, and these HMW multimers appear to be the more bioactive forms. Multimer composition of adiponectin appears to be regulated; however, the molecular mechanisms involved are unknown. We hypothesize that regulation of adiponectin multimerization and secretion occurs via changes in posttranslational modifications (PTMs). Although a structural role for intertrimer disulfide bonds in the formation of hexamers and HMW multimers is established, the role of other PTMs is unknown. PTMs identified in murine and bovine adiponectin include hydroxylation of multiple conserved proline and lysine residues and glycosylation of hydroxylysines. By mass spectrometry, we confirmed the presence of these PTMs in human adiponectin and identified three additional hydroxylations on Pro71, Pro76, and Pro95. We also investigated the role of the five modified lysines in multimer formation and secretion of recombinant human adiponectin expressed in mammalian cell lines. Mutation of modified lysines in the collagenous domain prevented formation of HMW multimers, whereas a pharmacological inhibitor of prolyl- and lysyl-hydroxylases, 2,2'-dipyridyl, inhibited formation of hexamers and HMW multimers. Bacterially expressed human adiponectin displayed a complete lack of differentially modified isoforms and failed to form bona fide trimers and larger multimers. Finally, glucose-induced increases in HMW multimer production from human adipose explants correlated with changes in the two-dimensional electrophoresis profile of adiponectin isoforms. Collectively, these data suggest that adiponectin multimer composition is affected by changes in PTM in response to physiological factors.

The isoflavonoids genistein and quercetin activate different stress signaling pathways as shown by analysis of site-specific phosphorylation of ATM, p53 and histone H2AX

The ataxia-telangiectasia mutated (ATM) protein kinase is activated in response to ionizing radiation (IR) and activates downstream DNA-damage signaling pathways. Although the role of ATM in the cellular response to ionizing radiation has been well characterized, its role in response to other DNA-damaging agents is less well defined. We previously showed that genistein, a naturally occurring isoflavonoid, induced increased ATM protein kinase activity, ATM-dependent phosphorylation of p53 on serine 15 and activation of the DNA-binding properties of p53. Here. we show that genistein also induces phosphorylation of p53 at serines 6, 9, 20,46, and 392, and that genistein-induced accumulation and phosphorylation of p53 is reduced in two ATM-deficient human cell lines. Also, we show that genistein induces phosphorylation of ATM on serine 1981 and phosphorylation of histone H2AX on serine 139. The related bioflavonoids, daidzein and biochanin A, did not induce either phosphorylation of p53 or ATM at these sites. Like genistein, quercetin induced phosphorylation of ATM on serine 198 1, and ATM-dependent phosphorylation of histone H2AX on serine 139; however, p53 accumulation and phosphorylation on serines 6, 9, 15, 20, 46, and 392 occurred in ATM-deficient cells, indicating that ATM is not required for quercetin-induced phosphorylation of p53. Our data suggest that genistein and quercetin induce different DNA-damage induced signaling pathways that, in the case of genistein, are highly ATM-dependent but, in the case of quercetin, may be ATM-dependent only for some downstream targets. (C) 2003 Elsevier B.V. All rights reserved.

Chemical and functional identification and characterization of novel sulfated alpha-conotoxins from the cone snail Conus anemone

An LC/MS analysis with diagnostic screening for the detection of peptides with posttranslational modifications revealed the presence of novel sulfated peptides within the -conotoxin molecular mass range in Conus anemone crude venom. A functional assay of the extract showed activity at several neuronal nicotinic acetylcholine receptors (nAChRs). Three sulfated alpha-conotoxins (AnIA, AnIB, and AnIC) were identified by LC/MS and assay-directed fractionation and sequenced after purification. The most active of these, alpha-AnIB, was further characterized and used to investigate the influence of posttranslational modifications on affinity. Synthetic AnIB exhibited subnanomolar potency at the rat alpha3/beta2 nAChR (IC50 0.3 nM) and was 200-fold less active on the rat alpha7 nAChR (IC50 76 nM). The unsulfated peptide [Tyr(16)]AnIB showed a 2-fold and 10-fold decrease in activities at alpha3beta2 (IC50 0.6 nM) and alpha7(IC50 836 nM) nAChR, respectively. Likewise, removal of the C-terminal amide had a greater influence on potency at the alpha7 (IC50 367 nM) than at the alpha3beta2 nAChR (IC50 0.5 nM). Stepwise removal of two N-terminal glycine residues revealed that these residues affect the binding kinetics of the peptide. Comparison with similar 4/7-alpha-conotoxin sequences suggests that residue 11 (alanine or glycine) and residue 14 (glutamine) constitute important determinants for alpha3beta2 selectivity, whereas the C-terminal amidation and sulfation at tyrosine-16 favor alpha7 affinity.

Comparative proteomic analysis to study molecular events during gonad development in mice

Sex determination represents a critical bifurcation in the road of embryonic development. It is based on a finely regulated network of gene activity, as well as protein-protein interactions and activation or silencing of signaling pathways. Despite the identification of a number of critical genes, many aspects of the molecular cascade that drives the differentiation of the embryonic gonad into either a testis or an ovary remain poorly understood. To identify new proteins involved in this cascade, we employed two-dimensional gel electrophoresis and mass spectrometry to compare the protein expression profiles of fetal mouse testes and ovaries. Three proteins, hnRPA1, TRA1, and HSC71, were found to be expressed in a male-specific manner and this expression was confirmed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and in situ hybridization. Moreover, HSC71 was found to be hyperphosphorylated in male compared to female gonads, emphasizing the advantage of the proteomic approach in allowing the detection of posttranslational modifications.

Responding to our customers' needs: modifications to assessment in Obstetrics and Gynaecology over 20 years

Background and Objectives: This paper reports on historical changes in assessment culminating in the experience of one discipline with negotiated student feedback that has helped design and modify assessment to cater for the requirements of both students and teachers. The standard of assessment required to pass Obstetrics and Gynaecology in the four year graduate entry program in the School of Medicine at The University of Queensland, Brisbane, Australia has become less formalised and more collaborative. Changes in assessment in this discipline over the last 20 years reflect the development of an understanding of the educational principles associated with adult teaching and learning. Assessment has evolved from being teacher focussed, with questionable reliability, validity, and emphasis on outcomes, to being focussed on learning and the student. Multiplechoice examinations, combined with a collaborative approach to the reliability and validity of questions and answers and a debrief or feedback session have been found to provide an assessment format that is art acceptable measure oflearning for both teachers and students. Changes in assessment reflect a collaborative process between teachers and students based on principles of adult learning and involving negotiated student feedback. Our experience with this form of negotiated outcome for assessment is presented together with suggestions for improvement and is contrasted with assessment methods used in this department over the last 20 years. Change and refinement will continue as medical programs strive to meet the learning needs of students and assessment outcomes that are acceptable to its teachers.

Coconut (Cocos nucifera) in vitro ecology: Modifications of headspace and medium additives can optimize somatic embryogenesis

Of those explants tested, immature zygotic embryo tissues proved to be the best for initiating callus with potential for somatic embryogenesis. Slicing of this tissue and use of the central sections (near to and including the meristematic tissue) gave the best embryogenic response. Slices that were placed under illumination necrosed more rapidly and to a greater degree than those incubated in the dark. Explant slice necrosis could be prevented or severely retarded by the addition of activated charcoal into the medium. Washing the explants for short periods of time prior to culture was also found to improve callus production. Prolonged washing resulted in low rates of callus production. In an attempt to prevent ethylene accumulation in the culture vessel headspace, AVG, an ethylene biosynthesis inhibitor and STS, a chemical which reduces the physiological action of ethylene, were successfully used to promote somatic embryogenesis. Spermidine, putrescine and spermine, polyamines that are known to delay plant senescence and promote somatic embryogenesis in some plant species, enhanced the rate of somatic embryogenesis when they were introduced into the callus induction medium. The use of polyethylene glycol in combination with abscisic acid helped promote somatic embryo formation and maturation as well as the subsequent formation of plantlets. The use of all of these improvements together has created a new and improved protocol for coconut somatic embryogenesis. This new protocol puts significant emphasis on improving the in vitro ecology of the explant, callus and somatic embryogenic tissues.

Norm modifications in defeasible logic

This paper proposes a framework based on Defeasible Logic (DL) to reason about normative modifications. We show how to express them in DL and how the logic deals with conflicts between temporalised normative modifications. Some comments will be given with regard to the phenomenon of retroactivity.