Common End Product Research Articles

Abstract 2330: MCT inhibition as a therapeutic strategy to target Enzalutamide-resistant prostate cancer

Abstract Sayani Bhattacharjee, Jonathan Doan, Rebecca Wynn, Nagalakshmi Nadiminty Enzalutamide is a second-generation anti-androgen used for the treatment of prostate cancer. However, almost all patients develop resistance to this drug, thereby creating an urgent need of identifying new approaches that can overcome said resistance. Using parental and Enzalutamide-resistant C4-2B and 22RV1 cells, we targeted cellular metabolism to beat therapeutic resistance. Prostate cancers are more heterogeneous in their use of energy sources compared with other solid tumors and the most common metabolic end product is lactate, which is toxic to cancer cells upon accumulation. Hence, cancer cells upregulate the expression of monocarboxylate transporters (MCTs) that play a pivotal role in lactate efflux. Different MCT isoforms are differentially expressed across prostate cancer progression and presumably according to cellular demands at different stages. Gradual progression from benign prostate, to prostatic intraepithelial neoplasia, to in-situ carcinoma showed an increase in the expression of MCT2 and MCT4, with MCT4 being expressed in invasive prostate cancers. Based on our preliminary data that the expression of MCTs is higher in prostate cancer cells resistant to enzalutamide, we hypothesized that MCT inhibition may be an attractive therapeutic strategy against enzalutamide-resistant prostate cancer cells. Using cell survival, cell proliferation and clonogenic-ability assays, we found that the suppression of MCT activity using the MCT1/2 antagonists AR-C155858 or AZD3965 and the MCT 4 antagonist Syrosingopine not only diminishes the proliferation and survival of prostate cancer cells, but also re-sensitizes resistant cells to treatment with enzalutamide. We also found that treatment with AR-C155858 or AZD3965 either singly, or in combination with Enzalutamide suppressed prostate cancer cell xenograft growth in SCID mice bearing subcutaneously injected parental or Enzalutamide-resistant C4-2B cells. These findings imply that the MCTs have an intrinsic role in the acquisition of resistance to therapy. Citation Format: MCT inhibition as a therapeutic strategy to target Enzalutamide-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2330.

Environmental Stability of Schwertmannite: A Review

Schwertmannite is sensitive to changes in geochemical, thermal, and microbial conditions. Changes in aqueous pH beyond its stability, i.e. pH 2.5–4.5, triggers its transformation to jarosite or goethite in highly acidic environments (pH ≤ 2.5), depending on the availability of jarosite-directing cations (Na+, NH4+, K+, etc.), while goethite is the common stable end product at pH > 7.5. Schwertmannite with degraded morphology can stably exist for years in oxic intermediate pH environments (pH 5.5–6.5), but the presence of trace amounts of Fe(II)aq yields goethite/lepidocrocite within a few hours, especially at pH ≥ 6.5. Hematite is the sole end product at ≥ 600 °C dry heating, with goethite and ferrihydrite as intermediate phases. Siderite, maghemite, and mackinawite form in anoxic microbial conditions due to dissimilatory reduction of Fe(III) and SO42− to Fe(II) and HS−, while orpiment forms from As(V)-rich schwertmannites. Sorbed contaminants enhance schwertmannite stability by restricting Fe(II)–Fe(III) electron transfer and microbial degradation by occupying surface sites. Although Fe(III) and sorbed ion mobilization typically has negligible effects on schwertmannite transformation, complete schwertmannite-SO4 release is likely in extreme conditions, and in microbial Fe(II)aq-rich media. Dissolution–reprecipitation and solid state transformation mechanisms broadly govern schwertmannite transformation.

Liquid chromatography-tandem mass spectrometry is effective for analysis of ergosterol in fungal-infected nails.

Identification of onychomycosis is mainly based on clinical diagnosis with auxiliary diagnostic methods such as potassium hydroxide (KOH) microscopy, periodic acid-Schiff staining or fungal culture. However, each method is limited by its sensitivity and specificity. To develop a new test method using the common fungal end product, ergosterol, and investigate if it can be used as a new diagnostic tool. We collected consecutive data from 20 participants with nail problems. Following clinical diagnosis, samples were taken for KOH microscopy and for mass spectrometry (MS) to check for the presence of ergosterol. Of the 20 cases collected, 7 were positive for fungal infection by MS. Four of these were already suspected to have onychomycosis, whereas one of the remaining three subjects was presumed to have dry nail and the other two to have onycholysis. The MS test seemed to be better at detecting combinations of nail conditions. Conversely, of the five patients clinically diagnosed as having onychomycosis, four had a positive MS result, whereas the fifth had negative results on both KOH and MS. Two other participants had a positive KOH test and were also found to have positive MS results. Detection of the presence of ergosterol by MS seems to be a useful tool for confirming onychomycosis. However, further studies are needed to verify the sensitivity and specificity of this MS method.

Management of stress exerted by hydrogen peroxide in Drosophila melanogaster using Abhrak bhasma

One of the undesirable outcomes of aerobic respiration is development of reactive oxygen species (ROS) and other free radicals. The cellular antioxidant system continuously reduces these into less toxic molecules. A common end product of quenching of ROS is hydrogen peroxide (H2O2), though this too is an oxidant and is further converted into water and molecular oxygen by antioxidant enzymes. We monitored selected indicators of oxidative stress after exposing Drosophila larvae to sub lethal concentration of H2O2. The efficacy of dietary supplementation of abhrak bhasma and ascorbic acid in relieving the oxidative stress, induced by H2O2, was also evaluated in this study. The exposure of Drosophila larvae to H2O2 was found to modulate the activity of SOD and catalase enzymes. The larvae exposed to H2O2 but maintained on a medium free of antioxidant supplement (control) exhibited decrease in total GSH content, GSH:GSSG ratio and capacity to scavenge DPPH free radicals. Overall, supplementing the diet with abhrak bhasma seems to enhance the antioxidant machinery of the Drosophila larvae. The comparable results were also obtained for the larvae maintained in a medium supplemented with a known antioxidant, ascorbic acid.

Association of Low Ficolin-Lectin Pathway Parameters with Cardiac Syndrome X.

In patients with typical angina pectoris, inducible myocardial ischaemia and macroscopically normal coronaries (cardiac syndrome X (CSX)), a significantly elevated plasma level of terminal complement complex (TCC), the common end product of complement activation, has been observed without accompanying activation of the classical or the alternative pathways. Therefore, our aim was to clarify the role of the ficolin-lectin pathway in CSX. Eighteen patients with CSX, 37 stable angina patients with significant coronary stenosis (CHD) and 54 healthy volunteers (HC) were enrolled. Serum levels of ficolin-2 and ficolin-3, ficolin-3/MASP-2 complex and ficolin-3-mediated TCC deposition (FCN3-TCC) were determined. Plasma level of TCC was significantly higher in the CSX than in the HC or CHD group (5.45 versus 1.30 versus 2.04AU/ml, P<0.001). Serum levels of ficolin-2 and ficolin-3 were significantly lower in the CSX compared to the HC or CHD group (3.60 versus 5.80 or 5.20μg/ml, P<0.05; 17.80 versus 24.10 or 26.80μg/ml, P<0.05). The ficolin-3/MASP-2 complex was significantly lower in the CSX group compared to the HC group (92.90 versus 144.90AU/ml, P=0.006). FCN3-TCC deposition was significantly lower in the CSX group compared to the HC and CHD groups (67.8% versus 143.3% or 159.7%, P<0.05). In the CSX group, a significant correlation was found between TCC and FCN3-TCC level (r=0.507, P=0.032) and between ficolin-3/MASP-2 complex level and FCN3-TCC deposition (r=0.651, P=0.003). In conclusion, in patients with typical angina and myocardial ischaemia despite macroscopically normal coronary arteries, low levels of several lectin pathway parameters were observed, indicating complement activation and consumption. Complement activation through the ficolin-lectin pathway might play a role in the complex pathomechanism of CSX.

Trehalose-6-phosphate synthase and stabilization of yeast glycolysis.

‘Lost in transition: Startup of glycolysis yields subpopulations of nongrowing cells…’ (‘LIT’, van Heerden et al . 2014) is a massive paper from groups in Amsterdam and Delft, which deals with broad issues in metabolism and cell heterogeneity, as addressed for the predominant metabolic pathway, glycolysis, in the context of a long studied but incompletely understood yeast mutant which is impaired in use of glucose without evident direct defects in the pathway. The primary approach is the quite original one of predicting, for the mutant, the dynamics of metabolism upon glucose addition, based on a mathematical model using the known kinetics for the enzymes of the pathway. Here we will discuss this paper and provide some additional model simulations illuminating the model. The stoichiometry of the glycolytic pathway using whole yeast cells and glucose can be near to 1 Glucose → 2 Ethanol + 2 CO2. Such a simple balance may be seen in fermentative circumstances throughout biology although lactic acid is a more common end product. The pathway, as worked out in extracts in the first decades of the 20th century turned out to involve 10 or so steps to pyruvic acid, using two ATP in the upper reactions and two inorganic phosphate (Pi) and four ADP in the lower reactions, for a net, per glucose, of two inorganic phosphate (Pi) + 2ADP → 2ATP + 2 H2O (as abbreviated in Fig. 1). In the cell extracts from yeast, however, at one time the stoichiometry appeared quite different from that in whole cells, with half of the glucose accumulating as the intermediate, fructose-1,6-P2 (Fru1,6P2) under certain conditions (Harden 1923). The discrepancy between in vivo and in vitro was explained in hindsight: ATP was not yet known and rapid flux requires its constant use. …

Review of Selected National Surgical Curricula: Quantity is Not the Sole Marker of Quality

Despite marked global variations in length and structure of surgical training programs, their common end product is a trained surgeon capable of independent practice. If variations exist, yet the end product is similar, modifications to curricula could potentially enhance the quality and efficiency of surgical training. This review evaluates global general surgery training programs and compares their curricula against the established standards for assessment of curricula. A convenience sampling method was employed during an online search for nationally recognized general surgery curricula. Curricula of Australia, Canada, Hong Kong, the United Kingdom, and the United States of America were individually reviewed and subsequently evaluated against the General Medical Council's "Standards for curricula and assessment systems." Postgraduate surgical training is completed in 5 years in Canada and the United States, whereas this takes a minimum of 7, 7, and 10 years in Australia, Hong Kong, and the United Kingdom, respectively. However, when their general surgery curricula are objectively compared, they are remarkably similar. The principle disparities noted were in documentation and standardization of the structured in-training assessment system. This review highlights variations in the structure of general surgery training programs globally. There is a need for an objective method to assess training quality, not reliant upon quantity alone. An evidence-based approach is the gold standard in patient care; it is essential to invest resources into developing an evidence-based curricular approach to ensure surgical training quality can be accurately evaluated to maintain and enhance the standards.

Enantioseparation and Absolute Configuration Determination of Angular-Type Pyranocoumarins from Peucedani Radix Using Enzymatic Hydrolysis and Chiral HPLC-MS/MS Analysis

Angular-type pyranocoumarins from Peucedani Radix (Chinese name: Qian-hu) have exhibited potential for use on treatment of cancer and pulmonary hypertension. Due to the existence of C-3′ and C-4′ chiral centers, compounds belonging to this chemical type commonly exist in enantiomers and/or diastereoisomers, which may elicit distinct activities during their interactions with the human body. In the present study, a new method, which combines enzymatic hydrolysis with chiral LC-MS/MS analysis, has been developed to determine the absolute configurations of these angular-type pyranocoumarins. Pyranocoumarins isolated from Qian-hu, their enantiomers, or metabolites were individually incubated with rat liver microsomes. As the common end product from enzymatic hydrolysis of all tested pyranocoumarins, cis-khellactone was collected and its absolute configuration was determined by comparison with (+)-cis-khellactone and (−)-cis-khellactone using chiral LC-MS/MS. The absolute configurations of all tested parent pyranocoumarins were determined by combination of LC-MS/MS, NMR and polarimetric analysis. The results revealed that the metabolite cis-khellactone retained the same absolute configurations of the stereogenic carbons as the respective parent compound. This method was proven to be rapid and sensitive and also has advantages in discriminating single enantiomers and mixtures of optical isomers with different ratios.

Seasonal variation in diet composition and similarity of sympatric red deer Cervus elaphus and roe deer Capreolus capreolus

Use of food resources by herbivores depends on intrinsic constraints, essentially body size and morpho-physiological characteristics, which determine the range of foods they tolerate and environmental constraints, such as seasonality and interspecific interactions, which determine the availability of resources. We analysed a collection of rumen contents samples from sympatric populations of red deer Cervus elaphus and roe deer Capreolus capreolus and tested several theoretical predictions relating to the impact of intrinsic and environmental constraints on diet composition, diversity and similarity. Red deer consumed more slowly digestible, fibrous forage than roe deer and had a more diverse diet throughout the year, which supports predictions deriving from specific body size and morpho-physiological characteristics. In conformity with the optimal foraging theory, both species consumed more slowly digestible forage in times of low food availability (i.e. during winter) than during the rest of the year. An increase in diet similarity in winter, along with predictions from the theory on competitive interaction processes, led us to assume that food resources were not limiting and that exploitative competition between red and roe deer was unlikely in our study area. We underline the importance of studies of the use of food resources by sympatric herbivores in answering applied ecological questions at the local scale, and we suggest that the Euclidean geometrical approach we used is particularly well suited for the analysis of resource matrices, a common end-product of long-term field data gathering on the feeding habits of animals.

Intermediates in the oxidative pathway from torulene to torularhodin in the red yeasts Cystofilobasidium infirmominiatum and C. capitatum (Heterobasidiomycetes, Fungi)

Two red Cystofilobasidium spp. isolated from spring sap-flows of Betula pendula were analysed for their carotenoid content. In Cystofilobasidium infirmominiatum, three unusual pigments were detected and identified by structure elucidation as oxidised torulene derivatives. These included 16′-hydroxytorulene and torularhodinaldehyde, two carotenoids known so far only from chemical synthesis or as postulated biosynthetic intermediates en route to torularhodin. Unprecedented formation of β-apo-2′-carotenal was also observed. The production of these pigments in pure culture was dependent on enhanced oxidative stress caused by cultivation in well-aerated (indented) flasks with or without 2% ethanol (16′-hydroxytorulene), or with 100 μM duroquinone (torularhodinaldehyde and β-apo-2′-carotenal). Among these three pigments, only 16′-hydroxytorulene was detected in C. capitatum. Torularhodin, a common end product of carotenoid oxidation in red yeasts, was not produced by either species under any incubation conditions. Biosynthetic aspects of incomplete oxidation of torulene by these Cystofilobasidium spp. are discussed.

Searching for non-invasive markers of tissue hypoxia

Tissue hypoxia is a common end product of circulatory shock and a primary target for resuscitation efforts. In this issue Podbregar and Mozina show that thenar tissue O2 saturation (StO2) and mixed venous O2 saturation (SvO2) co-vary in patients in left ventricular failure, but in patients with sepsis StO2 was higher than SvO2. Although StO2 may co-vary with SvO2 they have different determinants such that after shock StO2 may increase well before SvO2 as a result of increased O2 demands to repay O2 debt incurred during hypoperfusion. Thus, the use of StO2 alone to define the endpoint of resuscitation may be misleading.

Investigation of cu(I)-dependent 2,4,5-Trihydroxyphenylalanine Quinone Biogenesis in Hansenula polymorpha Amine Oxidase

Copper, a mediator of redox chemistries in biology, is often found in enzymes that bind and reduce dioxygen. Among these, the copper amine oxidases catalyze the oxidative deamination of primary amines utilizing a type(II) copper center and 2,4,5-trihydroxyphenylalanine quinone (TPQ), a covalent cofactor derived from the post-translational modification of an active site tyrosine. Previous studies established the dependence of TPQ biogenesis on Cu(II); however, the dependence of cofactor formation on the biologically relevant Cu(I) ion has remained untested. In this study, we demonstrate that the apoform of the Hansenula polymorpha amine oxidase readily binds Cu(I) under anaerobic conditions and produces the quinone cofactor at a rate of 0.28 h(-1) upon subsequent aeration to yield a mature enzyme with kinetic properties identical to the protein product of the Cu(II)-dependent reaction. Because of the change in magnetic properties associated with the oxidation of copper, electron paramagnetic resonance spectroscopy was employed to investigate the nature of the rate-limiting step of Cu(I)-dependent cofactor biogenesis. Upon aeration of the unprocessed enzyme prebound with Cu(I), an axial Cu(II) electron paramagnetic resonance signal was found to appear at a rate equivalent to that for the cofactor. These data provide strong evidence for a rate-limiting release of superoxide from a Cu(II)(O(2)(.)) complex as a prerequisite for the activation of the precursor tyrosine and its transformation for TPQ. As copper is trafficked to intracellular protein targets in the reduced, Cu(I) state, these studies offer possible clues as to the physiological significance of the acquisition of Cu(I) by nascent H. polymorpha amine oxidase.

Utility of Mass Spectrometry for In-Vitro ADME Assays

A balance between pharmacological activity, safety and drug metabolism and pharmacokinetics (DMPK) attributes determines the fate of a new chemical entity (NCE) in drug discovery. Because of the increased number of NCEs requiring DMPK evaluation, several in vitro higher-throughput screens and counter screens designed to evaluate DMPK attributes have been introduced in drug discovery. The DMPK screens evaluate NCEs for potential absorption, metabolism, drug-drug interactions, brain penetration, protein binding and pharmacokinetics. Higher-throughput analytical methodologies for the determination of either a common end product of a screen or the parent compound (and/or possible metabolites) are essential for successful DMPK screens. Because of its speed, sensitivity and specificity, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the technology of choice for sample analysis. In this review, several in vitro screening assays that we employ in drug discovery are discussed with an emphasis on LC-MS/MS role in accelerating them.

Degradation of Azo Dyes by Laccase and Ultrasound Treatment

The goal of this work was to investigate the decomposition of azo dyes by oxidative methods, such as laccase and ultrasound treatments. Each of these methods has strong and feeble sides. The laccase treatment showed high decolorization rates but cannot degrade all investigated dyes (reactive dyes), and high anionic strength led to enzyme deactivation. Ultrasound treatment can decolorize all tested dyes after 3 h at a high energy input, and prolonged sonication leads to nontoxic ionic species, which was demonstrated by ion chromatography and toxicity assays. For the first time, it was shown that a combination of laccase and ultrasound treatments can have synergistic effects, which was shown by higher degradation rates. Bulk light absorption and ion-pairing high-performance liquid chromatography (IP-HPLC) were used for process monitoring, while with reversed-phase HPLC, a lower number of intermediates than expected by IP-HPLC was found. Liquid chromatography-mass spectrometry indicated that both acid orange dyes lead to a common end product due to laccase treatment. Acid Orange 52 is demethylated by laccase and ultrasound treatment. Further results confirmed that the main effect of ultrasound is based on *OH attack on the dye molecules.

Photoluminescence Characterization of SiGe/Si Quantum-Well Wire Structures

ABSTRACTLow-temperature photoluminescence measurements were performed in order to probe the optical quality of SiGe/Si quantum-well wire structures fabricated by electron-beam lithography and subsequent reactive ion etching, having the patterned polymethylmethacrylate resist as an etch mask. In addition, one set of quantum-well wire structures was post-treated by means of annealing in a hydrogen environment. Our results show that even for the smallest wires of about 100nm in width, the wires exhibit phonon-resolved photoluminescence spectra, similar to that from the molecular beam eptitaxially grown SiGe single quantum well which was used as starting material for the patterning process. After the patterning process a new sharp peak appears in the photoluminescence spectra at 0.97eV in photon energy. Our investigation suggests that this feature is introduced by damage during the patterning process and most probably identical to the G-line, which previously was identified as originating from the dicarbon centre (substitutional carbon-interstitial carbon) in Si. This centre is known to be a very common endproduct of irradiating Si near room temperature which is the case at our patterning process.

Persistence and Movement of Sethoxydim Residues in Three Minnesota Soils

The persistence and movement of sethoxydim residues were determined in the top 45 cm of an Estherville sandy loam (sl), Port Byron silt loam (sil), and Webster clay loam (cl) in the field. Analysis of sethoxydim residues simultaneously quantified parent and eight metabolites by conversion to a common volatile end product, 3-[2(ethylsulfonyl)propyl]-pentanedioic acid dimethyl ester, followed by analysis using gas chromatography (GC) and a flame photometric detector. Recovery of residues from soil spiked with 100 μg kg-1was 86 ± 21%. Residues remaining in soil 21 days after treatment (DAT) in 1989 were 37, 51, and 29% of the applied sethoxydim in the sl, sil, and cl, respectively, while in 1990, 86, 52, and 24% remained in the sl, sil, and cl, respectively. Alachlor remaining 21 DAT in 1989 was 66, 52, and 65% of that applied in the sl, sil, and cl, respectively, while in 1990, it was 16, 13, and 100% in the sl, sil, and cl, respectively. Atrazine had the greatest % of applied chemical remaining 21 DAT in 1989: 100, 71, and 73% in the sl, sil, and cl, respectively. of the atrazine applied in 1990, atrazine remaining 21 DAT was 87% in the sl, 42% in the sil, and 100% in the cl. Over all soils and years, the amount of total sethoxydim residues remaining 137 DAT was the least of the three herbicides. In terms of leaching, sethoxydim residues showed least movement, with minimal detections below 0 to 15 cm. Although sethoxydim appears to be advantageous over alachlor and atrazine in terms of potential impact on ground water quality, additional information regarding composition of the detected residues is needed to better assess its environmental impact.

The corrosion of uraninite under oxidizing conditions

Abstract Uraninite is the most common uranium mineral, which, because of chemical and structural similarities to synthetic UO 2, is a useful natural analogue for UO 2 in spent fuel. Uraninite, however, contains “impurities” such as Pb, Ca, Si, U 6+, Th, Zr, and lanthanides. These affect the thermodynamic properties of uraninite, the rate of uraninite alteration, and the composition of the corrosion products. Uraninite can contain a significant amount of radiogenic Pb, and the Pb-uranyl oxide hydrates (Pb-UOH) are the most common corrosion products formed by the oxidative alteration of Pb-bearing uraninites. Incongruent alteration of the Pb-UOHs in natural waters produces increasingly Pb-enriched uranyl phases, effectively reducing the amount of U lost from the corrosion rind. This is not true of other uranyl oxide hydrates, such as schoepite, UO 3 · H 2O, or becquerelite, CaU 6O 19 · 11H 2O, which can dissolve completely under similar geochemical conditions. The most common end product of Pb-UOH alteration is curite. Curite may provide surface nucleation sites for certain uranyl phosphates, thereby enhancing their formation. Uranyl phosphates are generally less soluble than other uranyl phases. In the absence of Pb, schoepite and becquerelite are the common initial corrosion products. The reaction path for the alteration of Pb-free uraninite results in the formation of uranyl silicates, which are generally more soluble than the uranyl phosphates. Thus, the long-term oxidation behavior for ancient, Pb-bearing uraninite is different from young, Pb-free uraninite. Because the presence of Pb effectively reduces the mobility of uranium in oxidizing waters, the concentration of uranium in ground waters associated with oxidized uranium ore deposits will depend in part on the age of the primary uraninite.

N-acetyl-S-(2-hydroxyethyl)-L-cysteine as a potential tool in biological monitoring studies?

In mammalian species, including man, N-acetyl-S-(2-hydroxyethyl)-L-cysteine (2-HEMA) is a common urinary metabolite of a large number of structurally different xenobiotic chemicals. It is a common urinary end product of glutathione pathway metabolism of a variety of chemicals possessing electrophilic properties and, in most cases, also a genotoxic potential. Five different chemically reactive intermediates, with different electrophilic properties, may be involved in the formation of 2-HEMA. An inventory of chemicals known to lead to the formation of 2-HEMA, or based on their chemical structure expected to do so, is presented. Furthermore, an attempt is made to evaluate the possibilities and limitations in terms of the potential use of urinary 2-HEMA as a tool in biomonitoring studies. Two other related, sulfur-containing urinary metabolites, i.e. N-acetyl-(S-carboxymethyl)-L-cysteine and thio-diacetic acid, are proposed as possible alternatives to urinary 2-HEMA. It is suggested that 2-HEMA might be seen as a potentially useful and sensitive signal parameter for the assessment of exposure of animals and man to a variety of electrophilic and therefore potentially toxic xenobiotic chemicals.

Mechanism of protein carboxyl methyl transfer reactions: structural requirements of methyl accepting substrates.

The enzyme S-adenosylmethionine: protein carboxyl-O-methyltransfe-rase (PCMT) was first identified in 1965 by Axelrod and Daly as “methanol forming “ enzyme, since it was thought to catalyze the hydrolysis of S-adenosylmethionine (AdoMet) yielding methanol and S-adenosylhomocysteine (AdoHcy) (1). A few years later Liss et al. (2) and Kim and Paik (3) described an AdoMet-dependent methyl transferase which methylates free carboxyl groups in protein substrates and leads to the formation of unstable methyl esters. The two enzymes shared methanol as a common end product and were found to be identical on the basis of their properties and tissue distribution (4). During the following fifteen years the enzyme was purified from eukaryotic and prokaryotic sources, extensively characterized, and a number of different regulatory roles have been attributed to it, as reviewed by Paik and Kim in 1980 (5). However, in eukaryotes none of the hypothesized regulatory functions has been unequivocally demonstrated. More recently the eukaryotic and prokaryotic enzymes were found to differ in terms of methylation site, substrate specificity and functional role (6,7); therefore a new systematic name and classification number have been assigned to the eukaryotic PCMT. This enzyme indeed has been shown to recognize and selectively modify proteins at D-aspartyl and L-isoaspartyl residue levels (6,7).KeywordsDisulfide BridgeMethyl EsterificationIsopeptide BondErythrocyte Membrane ProteinNormal PeptideThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Hexuronate catabolism in Erwinia chrysanthemi

In the phytopathogenic enterobacterium Erwinia chrysanthemi, the catabolism of hexuronates is linked to the degradation of pectic polymers. We isolated Mu lac insertions in each gene of the hexuronate pathway and used genetic fusions with lacZ (the beta-galactosidase gene of Escherichia coli) to study the regulation of this pathway. Three independent regulatory genes (exuR, uxuR, and kdgR) were found. Galacturonate and glucuronate were converted into 2-keto-3-deoxygluconate (KDG) by separate three-step pathways encoded by the uxaC, uxaB, and uxaA genes and the uxaC, uxuB, and uxuA genes, respectively. The two aldohexuronates entered the cell by a specific transport system, encoded by exuT. Wild-type strain 3937 was unable to use glucuronate as a carbon source since glucuronate was unable to induce the exuT expression. Mutants able to use glucuronate possessed an inactivated exuR gene. The product of the regulatory gene exuR negatively controlled the expression of exuT, uxaC, uxaB, and uxaA, which was inducible in the presence of galacturonate. The two genes specifically involved in glucuronate catabolism, uxuA and uxuB, formed two independent transcriptional units regulated separately, uxuB expression was not inducible, whereas uxuA expression was induced in the presence of glucuronate and controlled by the uxuR product. KDG, the common end product of both pathways, is cleaved by the kdgK and kdgA gene products. KDG enters the cell by a specific transport system, encoded by kdgT. The regulatory gene kdgR controlled the expression of kdgT, kdgK, and kdgA and partially that of the pel genes encoding pectate-lyases. The real inducer of pectate-lyase synthesis, originating from catabolism of galacturonate or glucuronate, appeared to be KDG. The genes of E. chrysanthemi affecting hexuronate catabolism are separated into six independent transcriptional units exuT, uxaCBA, uxuA, uxuB, kdgK, and kdgA, but only three gene clusters were localized on the genetic map: exuT-uxaCBA, uxuA-uxuB-kdgK, and kdgA-exuR.