Phenolic compounds are well-known phytochemicals found in all plants. They consist of simple phenols, benzoic and cinnamic acid, coumarins, tannins, lignins, lignans and flavonoids. Substantial developments in research focused on the extraction, identification and quantification of phenolic compounds as medicinal and/or dietary molecules have occurred over the last 25 years. Organic solvent extraction is the main method used to extract phenolics. Chemical procedures are used to detect the presence of total phenolics, while spectrophotometric and chromatographic techniques are utilized to identify and quantify individual phenolic compounds. This review addresses the application of different methodologies utilized in the analysis of phenolic compounds in plant-based products, including recent technical developments in the quantification of phenolics.

Although the results fall just short of the specified noninferiority margin, the omission of bleomycin from the ABVD regimen after negative findings on interim PET resulted in a lower incidence of pulmonary toxic effects than with continued ABVD but not significantly lower efficacy. (Funded by Cancer Research UK and Others; ClinicalTrials.gov number, NCT00678327.).

Control of proteolysis is important for plant growth, development, responses to stress, and defence against insects and pathogens. Members of the serpin protein family are likely to play a critical role in this control through irreversible inhibition of endogenous and exogenous target proteinases. Serpins have been found in diverse species of the plant kingdom and represent a distinct clade among serpins in multicellular organisms. Serpins are also found in green algae, but the evolutionary relationship between these serpins and those of plants remains unknown. Plant serpins are potent inhibitors of mammalian serine proteinases of the chymotrypsin family in vitro but, intriguingly, plants and green algae lack endogenous members of this proteinase family, the most common targets for animal serpins. An Arabidopsis serpin with a conserved reactive centre is now known to be capable of inhibiting an endogenous cysteine proteinase. Here, knowledge of plant serpins in terms of sequence diversity, inhibitory specificity, gene expression and function is reviewed. This was advanced through a phylogenetic analysis of amino acid sequences of expressed plant serpins, delineation of plant serpin gene structures and prediction of inhibitory specificities based on identification of reactive centres. The review is intended to encourage elucidation of plant serpin functions.

Genes encoding proteins of the serpin superfamily are widespread in the plant kingdom, but the properties of very few plant serpins have been studied, and physiological functions have not been elucidated. Six distinct serpins have been identified in grains of hexaploid bread wheat (Triticum aestivum L.) by partial purification and amino acid sequencing. The reactive centers of all but one of the serpins resemble the glutamine-rich repetitive sequences in prolamin storage proteins of wheat grain. Five of the serpins, classified into two protein Z subfamilies, WSZ1 and WSZ2, have been cloned, expressed in Escherichia coli, and purified. Inhibitory specificity toward 17 proteinases of mammalian, plant, and microbial origin was studied. All five serpins were suicide substrate inhibitors of chymotrypsin and cathepsin G. WSZ1a and WSZ1b inhibited at the unusual reactive center P 1 -P 1 Gln-Gln, and WSZ2b at P 2 -P 1 LeuArg-one of two overlapping reactive centers. WSZ1c with P 1 -P 1 Leu-Gln was the fastest inhibitor of chymotrypsin (k a ‫؍‬ 1 The serpins constitute a superfamily of versatile proteins participating in the regulation of complex proteolytic systems (1, 2). Most serpins are serine proteinase inhibitors of chymotrypsin-like enzymes, but a few have been found to inhibit cysteine proteinases (3), and some are non-inhibitory. Many functionally distinct serpins have been identified in higher eukaryotes, some in viruses, but none in yeast or bacteria (4, 5).Mammalian serpins have been shown to participate in a growing number of extra-and intracellular physiological processes, including blood coagulation, complement activation, remodeling of the extracellular matrix, and hormone transport.The only plant serpin characterized in detail with respect to inhibitory specificity is recombinant barley (Hordeum vulgare) serpin rBSZx 1 (6 -8), but BSZx (GenBank™ accession number X97636) has not been detected in the plant. In addition, two serpins from barley grain, BSZ4 (7, 9 -11) and BSZ7 (GenBank™ accession number CAA64599) (12, 13), and one from wheat (Triticum aestivum) grain (7, 14, 15) have been studied. Despite the high concentration of these inhibitors in the grains of monocot cereals (up to 4% total protein), the physiological functions of plant serpins remain unknown. Increasing numbers of putative serpin genes and serpin mRNAs have been identified from other monocot plants, including rice and wild oats, from eudicot plants, including tomato, cotton, and the model plant Arabidopsis thaliana, and from the non-vascular plant Physcomitrella patens (EMBL/GenBank), suggesting that serpins are widespread in the plant kingdom.Inhibitory serpins are metastable proteins (Ͼ40 kDa) employing a unique "suicide substrate" mechanism of irreversible inhibition very different from the reversible "standard mechanism" used by other proteinase inhibitors (Ͻ25 kDa) (1). The serpin in its native, active conformation has a flexible reactive center loop (RCL) located near the C terminus and protruding from the main body of the protein...

Many enzymes of the bacteriochlorophyll and chlorophyll biosynthesis pathways have been conserved throughout evolution, but the molecular mechanisms of the key steps remain unclear. The magnesium chelatase reaction is one of these steps, and it requires the proteins BchI, BchD, and BchH to catalyze the insertion of Mg 2؉ into protoporphyrin IX upon ATP hydrolysis. Structural analyses have shown that BchI forms hexamers and belongs to the ATPases associated with various cellular activities (AAA ؉ ) family of proteins. AAA ؉ proteins are Mg 2؉ -dependent ATPases that normally form oligomeric ring structures in the presence of ATP. By using ATPase-deficient BchI subunits, we demonstrate that binding of ATP is sufficient to form BchI oligomers. Further, ATPase-deficient BchI proteins can form mixed oligomers with WT BchI. The formation of BchI oligomers is not sufficient for magnesium chelatase activity when combined with BchD and BchH. Combining WT BchI with ATPase-deficient BchI in an assay disrupts the chelatase reaction, but the presence of deficient BchI does not inhibit ATPase activity of the WT BchI. Thus, the ATPase of every WT segment of the hexamer is autonomous, but all segments of the hexamer must be capable of ATP hydrolysis for magnesium chelatase activity. We suggest that ATP hydrolysis of each BchI within the hexamer causes a conformational change of the hexamer as a whole. However, hexamers containing ATPase-deficient BchI are unable to perform this ATP-dependent conformational change, and the magnesium chelatase reaction is stalled in an early stage.ATPase ͉ chlorophyll biosynthesis ͉ integrin ͉ metallation ͉ sensor arginine

Key Points• PET-CT is the modern standard for staging Hodgkin lymphoma and can replace contrast enhanced CT in the vast majority of cases.• Agreement between expert and local readers is sufficient for the Deauville criteria to assess response in clinical trials and the community.International guidelines recommend that positron emission tomography-computed tomography (PET-CT) should replace CT in Hodgkin lymphoma (HL). The aims of this study were to compare PET-CT with CT for staging and measure agreement between expert and local readers, using a 5-point scale (Deauville criteria), to adapt treatment in a clinical trial: Response-Adapted Therapy in Advanced Hodgkin Lymphoma (RATHL). Patients were staged using clinical assessment, CT, and bone marrow biopsy (RATHL stage). PET-CT was performed at baseline (PET0) and after 2 chemotherapy cycles (PET2) in a response-adapted design. PET-CT was reported centrally by experts at 5 national core laboratories. Local readers optionally scored PET2 scans. The RATHL and PET-CT stages were compared. Agreement among experts and between expert and local readers was measured. RATHL and PET0 stage were concordant in 938 (80%) patients. PET-CT upstaged 159 (14%) and downstaged 74 (6%) patients. Upstaging by extranodal disease in bone marrow (92), lung (11), or multiple sites (12) on PET-CT accounted for most discrepancies. Follow-up of discrepant findings confirmed the PET characterization of lesions in the vast majority. Five patients were upstaged by marrow biopsy and 7 by contrast-enhanced CT in the bowel and/or liver or spleen. PET2 agreement among experts (140 scans) with a k (95% confidence interval) of 0.84 (0.76-0.91) was very good and between experts and local readers (300 scans) at 0.77 (0.68-0.86) was good. These results confirm PET-CT as the modern standard for staging HL and that response assessment using Deauville criteria is robust, enabling translation of RATHL results into clinical practice. (Blood. 2016;127(12):1531-1538

In animals, protease inhibitors of the serpin family are associated with many physiological processes, including blood coagulation and innate immunity. Serpins feature a reactive center loop (RCL), which displays a protease target sequence as a bait. RCL cleavage results in an irreversible, covalent serpin-protease complex. AtSerpin1 is an Arabidopsis protease inhibitor that is expressed ubiquitously throughout the plant. The x-ray crystal structure of recombinant AtSerpin1 in its native stressed conformation was determined at 2.2 Å . The electrostatic surface potential below the RCL was found to be highly positive, whereas the breach region critical for RCL insertion is an unusually open structure. AtSerpin1 accumulates in plants as a fulllength and a cleaved form. Fractionation of seedling extracts by nonreducing SDS-PAGE revealed the presence of an additional slower migrating complex that was absent when leaves were treated with the specific cysteine protease inhibitor L-trans-epoxysuccinyl-L-leucylamido (4-guanidino)butane. Significantly, RESPONSIVE TO DESICCATION-21 (RD21) was the major protease labeled with the L-trans-epoxysuccinyl-L-leucylamido (4-guanidino)butane derivative DCG-04 in wild type extracts but not in extracts of mutant plants constitutively overexpressing AtSerpin1, indicating competition. Fractionation by nonreducing SDS-PAGE followed by immunoblotting with RD21-specific antibody revealed that the protease accumulated both as a free enzyme and in a complex with AtSerpin1. Importantly, both RD21 and AtSerpin1 knock-out mutants lacked the serpin-protease complex. The results establish that the major Arabidopsis plant serpin interacts with RD21. This is the first report of the structure and in vivo interaction of a plant serpin with its target protease.Protease cascades are prominent mediators of rapid physiological responses in animals, playing a role in cellular immunity, blood clotting, and development. The proteolytic specificity of the serine and cysteine proteases involved dictates the fidelity of these reactions. The serpins are an important group of proteins that curb the activity of these cascades through specific irreversible inhibition of the proteases. For example, in Drosophila, the necrotic (nec) gene encodes a protease inhibitor of the serpin family. Necrotic protein controls a proteolytic cascade that activates the innate immune response to fungal and Gram-positive bacterial infections (1). In nec null mutants, Toll-mediated immune responses are constitutively activated, even in the absence of infection, implying that Nec continually restrains this immune response. As opposed to other types of protease inhibitors, serpins offer both an irreversible and tunable type of inhibition (reviewed in Ref.2). In their native conformation, serpins are in a stressed (spring-loaded) state with a solvent-exposed reactive center loop (RCL).3 Specific residues of the RCL are precisely accommodated by the target protease active site. Upon cleavage of the serpin peptide bond linking the P1 and ...

Oxygen deprivation limits the energy available for cellular processes and yet no comprehensive ATP budget has been reported for any plant species under O2 deprivation, including Oryza sativa. Using 3-d-old coleoptiles of a cultivar of O. sativa tolerant to flooding at germination, (i) rates of ATP regeneration in coleoptiles grown under normoxia (aerated solution), hypoxia (3% O2), and anoxia (N2) and (ii) rates of synthesis of proteins, lipids, nucleic acids, and cell walls, as well as K+ transport, were determined. Based on published bioenergetics data, the cost of synthesizing each class of polymer and the proportion of available ATP allocated to each process were then compared. Protein synthesis consumed the largest proportion of ATP synthesized under all three oxygen regimes, with the proportion of ATP allocated to protein synthesis in anoxia (52%) more than double that in normoxic coleoptiles (19%). Energy allocation to cell wall synthesis was undiminished in hypoxia, consistent with preferential elongation typical of submerged coleoptiles. Lipid synthesis was also conserved strongly in O2 deficits, suggesting that membrane integrity was maintained under anoxia, thus allowing K+ to be retained within coleoptile cells. Rates of protein synthesis in coleoptiles from rice cultivars with contrasting tolerance to oxygen deficits (including mutants deficient in fermentative enzymes) confirmed that synthesis and turnover of proteins always accounted for most of the ATP consumed under anoxia. It is concluded that successful establishment of rice seedlings under water is largely due to the capacity of coleoptiles to allocate energy to vital processes, particularly protein synthesis.