Two approaches for metabolic pathway analysis?

Metabolic pathway analysis is becoming increasingly important for assessing inherent network properties in (reconstructed) biochemical reaction networks. Of the two most promising concepts for pathway analysis, one relies on elementary flux modes and the other on extreme pathways. These concepts are closely related because extreme pathways are a subset of elementary modes. Here, the common features, differences and applicability of these concepts are discussed. Assessing metabolic systems by the set of extreme pathways can, in general, give misleading results owing to the exclusion of possibly important routes. However, in certain network topologies, the sets of elementary modes and extreme pathways coincide. This is quite often the case in realistic applications. In our opinion, the unification of both approaches into one common framework for metabolic pathway analysis is necessary and achievable.     

A New Similarity Between Two Clustering Solutions

InclUSteranalysis,howtoevaluatethesiededtybetWeentWoactionsisOfmuchhippo~e.Withpropersindhatymeasure,wemayhapetocomparetwoden~ofsamesetofobjectstostudytheeffeCtofUSingdifferentclusteringalgorithms(Gordon,1981).WeareabletocomparetheclusterresultswiththenaturalconfigUlationspraiedinthedata(Rand,1971).~aam~OfallutionsOfsimilardatacanidentifytheialluentialfactorsorobjects(Jolliffe,1988,1992).TheexistingmeadscanbeeasilydividedintotWot~.OneisthecorrelationbetweentwomergingordersOfhierarchical…     

Two of these or two of those?

Symmetrically dividing neuroepithelial cells may produce two daughters that are both proliferating or both postmitotic, as highlighted by Zigman et al. in this issue of Neuron and Sanada and Tsai in a recent issue of Cell. Here, I will attempt to offer a simple explanation why these results may be so different.     

Two complexes at the site of microtubule nucleation

<正>Cortical microtubule(MT)arrays are dynamic filamentous structures that are essential for cell differentiation and development in plants.However,the molecular mechanisms that control the organization of cortical MT arrays are not well understood.Early studies have revealed that the formation of cortical MT arrays involves MT nucleation on existing cortical MTs.The growth of new MTs follows the polarity of existing MTs and the orientation of new MTs is either in parallel with extant MTs or at a small angle(about40 degree)to the extant MTs[1].Nucleation machinery appears to be conserved between animals and plants in     

Two new“Meganthropus” mandibles from Java

There are now eleven known mandibular pieces from the Lower and Middle Pleistocene of Java, all but one being from the Sangiran site. All of these have been assigned toHomo erectus by most authorities, while others have suggested as many as four different hominoid taxa. Two of the mandibles, Sangiran 33 (Mandible H) and“Meganthropus”D (no Sangiran number yet assigned), are described here for the first time. The two new mandibles come from the Upper Pucangan Formation and date approximately 1.2–1.4 Myr. They are morphologically compatible with other“Meganthropus” mandibles described from Java. Despite attempts by numerous authorities to place all the Sangiran hominid mandibles in the species,H. erectus, the range of variation in metric and nonmetric features of the“Meganthropus” hominids is clearly beyond the know variation found inH. erectus. “Meganthropus” could represent a speciation from the well-knownH. erectus.     

Two forms of α-glucosidase from sugar-beet seeds

Two forms of -glucosidase (EC 3.2.1.20), designated as I and II, have been isolated from sugarbeet (Beta vulgaris L.) seeds by a procedure including fractionation with ammonium sulfate and ethanol, carboxymethyl-cellulose column chromatography, and preparative disc gel electrophoresis. The two enzymes were homogeneous by polyacrylamide disc gel electrophoresis. Their molecular weights were 98,000 (I) and 60,000 (II). -Glucosidase I readily hydrolyzed maltose, isomaltose, kojibiose, maltotriose, panose, amylose, soluble starch, amylopectin and glycogen. -Glucosidase II also hydrolyzed maltose, kojibiose and maltotriose but hydrolyzed the other substrates only very weakly or not at all. -Glucosidase I hydrolyzed soluble starch at a faster rate than maltose. It produced isomaltose and panose as the main -glucosyltransfer products from maltose, whereas maltotriose was the main product of -glucosidase II. -Glucosidase I hydrolyzed amylose liberating -glucose. The neutral-sugar content was calculated to be 2.7% for -glucosidase I and 8.8% for -glucosidase II. The main neutral sugar was mannose in -glucosidase I, and glucose in -glucosidase II.     

Two continuous coupled assays for ornithine-δ-aminotransferase

We have developed two new continuous coupled assays for ornithine-δ-aminotransferase (OAT) that are more sensitive than previous methods, measure activity in real time, and can be carried out in multiwell plates for convenience and high throughput. The first assay is based on the reduction of Δ¹-pyrroline-5-carboxylate (P5C), generated from ornithine by OAT, using human pyrroline 5-carboxylate reductase 1 (PYCR1), which results in the concomitant oxidation of NADH (nicotinamide adenine dinucleotide, reduced form) to NAD⁺ (nicotinamide adenine dinucleotide, oxidized form). This procedure was found to be three times more sensitive than previous methods and is suitable for the study of small molecules as inhibitors or inactivators of OAT or as a method to determine OAT activity in unknown samples. The second method involves the detection of l-glutamate, produced during the regeneration of the cofactor pyridoxal 5’-phosphate (PLP) of OAT by an unamplified modification of the commercially available Amplex Red l-glutamate detection kit (Life Technologies). This assay is recommended for the determination of the substrate activity of small molecules against OAT; measuring the transformation of l-ornithine at high concentrations by this assay is complicated by the fact that it also acts as a substrate for the l-glutamate oxidase (GluOx) reporter enzyme.     

Two Cys or Not Two Cys? That Is the Question; Alternative Oxidase in the Thermogenic Plant Sacred Lotus

Sacred lotus (Nelumbo nucifera) regulates temperature in its floral chamber to 32°C to 35°C across ambient temperatures of 8°C to 40°C with heating achieved through high alternative pathway fluxes. In most alternative oxidase (AOX) isoforms, two cysteine residues, Cys₁ and Cys₂, are highly conserved and play a role in posttranslational regulation of AOX. Further control occurs via interaction of reduced Cys₁ with α-keto acids, such as pyruvate. Here, we report on the in vitro regulation of AOX isolated from thermogenic receptacle tissues of sacred lotus. AOX protein was mostly present in the reduced form, and only a small fraction could be oxidized with diamide. Cyanide-resistant respiration in isolated mitochondria was stimulated 4-fold by succinate but not pyruvate or glyoxylate. Insensitivity of the alternative pathway of respiration to pyruvate and the inability of AOX protein to be oxidized by diamide suggested that AOX in these tissues may lack Cys₁. Subsequently, we isolated two novel cDNAs for AOX from thermogenic tissues of sacred lotus, designated as NnAOX1a and NnAOX1b. Deduced amino acid sequences of both confirmed that Cys₁ had been replaced by serine; however, Cys₂ was present. This contrasts with AOXs from thermogenic Aroids, which contain both Cys₁ and Cys₂. An additional cysteine was present at position 193 in NnAOX1b. The significance of the sequence data for regulation of the AOX protein in thermogenic sacred lotus is discussed and compared with AOXs from other thermogenic and nonthermogenic species.

Thermogenesis in Sacred Lotus

Sacred lotus (Nelumbo nucifera) is a thermogenic plant that regulates the temperature of its floral chamber between 32°C and 35°C for up to 4 d (Seymour and Schultze-Motel, 1996). Heating of plant tissues has been described as an adaptation to attract insect pollinators either by volatilization of scent compounds (Meeuse, 1975) or by providing a heat reward (Seymour et al., 1983), protect floral parts from low temperatures (Knutson, 1974), or provide the optimum temperature for floral development (Ervik and Barfod, 1999; Seymour et al., 2009). In sacred lotus, heat is produced by high rates of alternative pathway respiration (Watling et al., 2006; Grant et al., 2008); however, the mechanisms of heat regulation, which likely occur at a cellular level, remain unclear.

Alternative Oxidase

Alternative pathway respiration is catalyzed by the alternative oxidase protein (AOX), which acts as a terminal oxidase in the electron transport chain but, unlike the energy conserving cytochrome pathway (COX), complexes III and IV are bypassed and energy is released as heat. Traditionally, AOX activity was measured using oxygen consumption of tissue, cells, or isolated mitochondria in the presence or absence of AOX and COX inhibitors. However, this method does not accurately measure activity in vivo but does indicate the capacity of the alternative pathway (Ribas-Carbo et al., 1995; Day et al., 1996). The only method to date to accurately determine AOX activity, that is, flux of electrons through the AOX pathway in vivo, is to use oxygen isotope discrimination techniques (for review, see Robinson et al., 1995). Determining AOX activity in vivo is important because heat production in plants could be due to activity of either the AOX and/or plant uncoupling proteins. Using oxygen fractionation techniques, we have shown that flux through the AOX pathway is responsible for heating in sacred lotus (Watling et al., 2006; Grant et al., 2008). Furthermore, we were unable to detect any uncoupling protein in these tissues (Grant et al., 2008). AOX protein content within the sacred lotus receptacle increases markedly prior to thermogenesis, but it remains constant during heating (Grant et al., 2008), suggesting that regulation of heating occurs through posttranslational modification of the protein.

Posttranslational Regulation of AOX Protein

The plant AOX is a cyanide-insensitive dimeric protein located in the inner mitochondrial membrane (Day and Wiskich, 1995). The dimer subunits (monomers) can be linked via a noncovalent association (reduced protein) or covalently through the formation of a disulfide bridge (oxidized protein; Umbach and Siedow, 1993). The reduced protein when run on SDS-PAGE has a molecular mass of approximately 30 to 35 kD and the oxidized protein 60 to 71 kD; this holds true for AOX from a number of species, including soybean (Glycine max) roots and cotyledons (Umbach and Siedow, 1993), tobacco (Nicotiana tabacum) leaf (Day and Wiskich, 1995), and the thermogenic spadix of Arum maculatum (Hoefnagel and Wiskich, 1998).Regulation of AOX has been well studied in nonthermogenic plant species, and two mechanisms have been identified. Most AOX isoforms have two highly conserved Cys residues, Cys₁ and Cys₂ (defined in Berthold et al., 2000 and Holtzapffel et al., 2003), located near the N-terminal hydrophilic domain of the protein. In these isoforms, Cys₁ can either be reduced on both subunits of the AOX dimer, or the Cys₁ sulfhydryl groups can be oxidized to form a disulfide bridge (Rhoads et al., 1998). Reduction/oxidation modulation of AOX in vitro can be achieved using the sulfhydryl reductant dithiothreitol (DTT) to reduce the protein or diamide to oxidize the Cys residues. The reduced dimer can be further activated via the interaction of Cys₁ with α-keto acids, principally pyruvate (Rhoads et al., 1998; see McDonald [2008] for a model of posttranslational regulation of AOX). In addition, Cys₂ may also be involved in regulating AOX activity through interaction with the α-keto acid glyoxylate (which can also stimulate activity at Cys₁; Umbach et al., 2002).Recently, however, AOX proteins with different regulatory properties have been reported. Naturally occurring AOX proteins without the two regulatory Cys residues have been identified and, along with site-directed mutagenesis studies, used to further elucidate the specific roles of Cys₁ and Cys₂. The LeAOX1b isoform from tomato (Lycopersicon esculentum), which has a Ser residue at the position of Cys₁ and thus does not form disulfide linked dimers, is also activated by succinate rather than pyruvate when expressed in Saccharomyces cerevisiae (Holtzapffel et al., 2003). In Arabidopsis (Arabidopsis thaliana), uncharged or hydrophobic amino acid substitutions of either Cys result in an inactive enzyme, while positively charged substitutions produce an enzyme with higher than wild type basal activity but that is insensitive to pyruvate or succinate (Umbach et al., 2002). Single substitutions at Cys₁ or Cys₂ have revealed that glyoxylate can activate AOX via both Cys residues, but only one is needed for glyoxylate stimulation (Umbach et al., 2002, 2006). Double substitution mutants were not stimulated by either pyruvate or glyoxylate (Umbach et al., 2006).Previously, we determined that thermogenesis via the AOX pathway in the sacred lotus receptacle is precisely regulated through changes in AOX flux rather than changes to protein content (Grant et al., 2008). In this study, we investigated the nature of this regulation in mitochondria isolated from heating receptacles. Our aim was to elucidate the reduction/oxidation behavior of the AOX protein and the mechanisms of activation of cyanide-resistant respiration in sacred lotus receptacles to provide insights into the mechanism(s) of heat regulation in this species. We further investigated AOX regulation by determining the amino acid sequence of two novel AOX genes isolated from thermogenic receptacle tissue of sacred lotus.