1,2,5-Trisubstituted 1,4-diazepine-3-one: A novel dipeptidomimetic molecular scaffold

Summary The continuing effort to transform bioactive peptides into non-peptide peptidomimetics of therapeutic potential requires a diversity of tools such as molecular scaffolds, pseudopeptide modifications, and conformation mimetics. To this end, a novel polyfunctional monoheterocyclic system, 1,2,5-trisubstituted hexahydro-3-oxo-1H-1,4-diazepine ring (DAP), was designed. The linear precursor for the DAP was generated through a reductive alkylation step including a modified side chain and an α-amino function of two amino acid derivatives. Structural analysis of model diastereomeric DAPs, employing¹H and¹³C NMR and computer simulation, revealed the conformational preferences of this system. The structural similarities to the 1,4-benzodiazepine, a common molecular scaffold for many non-peptidic peptidomimetic agents, and the pronounced dipeptidomimetic character of the DAP system offer a new powerful tool to medicinal chemists engaged in rational peptide-based drug design.     

Seed development and vivipary in Zea mays L.

Seed development was investigated in kernels of developing wild-type and viviparous (vp-1) Zea mays L. Embryos and endosperm of wild-type kernels began to dehydrate at approx. 35 d after pollination (DAP); viviparous embryos did not desiccate but accumulated fresh weight via coleoptile growth in the caryopses. Concentrations of endogenous abscisic acid (ABA) in the embryo were relatively high early in development, being approx. 150 ng·g^-1 fresh weight at 20 DAP. The ABA content declined thereafter, falling to approx. 50 ng·g^-1 at 30 DAP. Endosperm ABA content was always low, being less than 20 ng·g^-1. There were no differences between wild-type and vp-1 tissues. Immature kernels did not germinate when removed from the ear until late in development. The ability to germinate was correlated with decreasing moisture content in the endosperm at the time of removal; premature drying of immature kernels resulted in greatly increased germination following imbibition. Excised embryos germinated precociously when removed from the endosperm as early as 25 DAP. Such germination could be prevented by treatment with 10^-5 M ABA or by lowering the solute potential (_s) of the medium with 0.3 M mannitol. Treatment of excised embryos with ABA led to internal ABA concentrations comparable to those in embryos in which germination was inhibited in situ. Mannitol treatment did not have this effect, although water-deficit stress of excised embryos resulted in substantial ABA production. Germinated vp-1 embryos were less sensitive to growth inhibition by ABA or mannitol than germinating wild-type embryos. The vp-1 seedlings were not wilty and their transpiration rates were reduced in response to ABA or water shortage.Abbreviations and symbols ABA abscisic acid - DAP days after pollination - FW fresh weight - vp-1 viviparous genotype - _s solute potential     

Biosynthesis of (1,3)(1,4)-β-glucan and (1,3)-β-glucan in barley (Hordeum vulgare L.)

Mixed membrane preparations from the coleoptiles and first leaves of young barley (Hordeum vulgare L. cv. Triumph) plants catalysed the synthesis of 55% methanol-insoluble labelled material from UDP[U-¹⁴C]glucose, the main components of which were identified as (1,3)(1,4)-- and (1,3)--D-glucans. The membrane preparations also catalysed the transformation of UDP-glucose into labelled low-molecular-weight products, mainly glucose (by phosphatase action), glucose-1-phosphate (by phosphodiesterase action) and glyco(phospho)lipids (by glycosyltransferase action). The formation of (1,3)(1,4)--glucans, (1,3)--glucans, and the other reactions competing for UDP-glucose, were monitored simultaneously and quantitatively by a novel procedure based on enzymatic analysis, thin-layer chromatography and digital autoradiography. Thus it was possible (i) to optimise conditions to obtain (1,3)(1,4)--glucan synthesis or (1,3)--glucan synthesis in isolation, and (ii) to study the influence of temperature, pH, cofactors, substrate concentration etc. on the (1,3)(1,4) and (1,3)--glucan synthesis reactions even when both occurred together. The synthesis of both -glucans was optimal at 20°C. In Tris-HCl buffer, the pH optima for (1,3)(1,4)--glucan synthesis and (1,3)--glucan synthesis were pH 8.5 and pH 7.0, respectively. Both glucan-synthesis reactions required Mg²⁺: (1,3)--glucan synthesis was optimal at 2 mM, whereas (1,3)(1,4)--glucan synthesis continued to increase up to 200 mM Mg²⁺, when the ion was supplied as the sulphate. (1,3)--Glucan synthesis was Ca²⁺ dependent and this dependence could be abolished by proteinase treatment. The K _m with respect to UDP-glucose was 1.5 mM for (1,3)--glucan synthesis and approximately 1 mM for (1,3)(1,4)--glucan synthesis. The (1,3)(1,4)--glucan formed in vitro had the same ratio of trisaccharide to tetrasaccharide structural blocks irrespective of the experimental conditions used during the synthesis: its enzymatic fragmentation pattern was indistinguishable from that of barley endosperm (1,3)(1,4)--glucan. This indicates either a single synthase enzyme, which is responsible for the formation of both linkage types, or two enzymes which are very tightly coupled functionally.Abbreviations G4G4G3G Glc(1,4)Glc(1,4)Glc(1,3)Glc (-linked) - UDP-Glc uridine-5-diphosphate glucose We are grateful to the Commission of the European Communities for the award of Training Fellowships to Christine Vincent and Martin Becker.     

d-Xylanase produced by Schizophyllum radiatum

Summary d-Xylanase (1,4--xylan xylanohydrolase, EC 3.2.1.8) was obtained from mycelial submerged culture of the mushroom Schizophyllum radiatum, grown on wheat straw pretreated with steam explosion as the substrate. The enzyme was purified 192-fold (specific activity 455 IU mg^-1 protein), with 37% yield with respect to total d-xylanase activity. Polyacrylamide electrophoresis of the d-xylanase peak showed a single band of protein whose molecular weight, calculated by electrophoretic mobility, was 25 700. The enzyme exhibited maximum activity at pH 4.9 and 55°C. d-Xylanase was stable from pH 5.0 to 7.5; its half-life was 12 h at 45°C. The Michaelis constant was 9.5 mg ml^-1 and V _max 0.37 mole min^-1. End-product analysis of the d-xylan hydrolysate showed the presence of d-xylobiose, d-xylotriose, d-xylotetraose, and d-xylopentose showing the mode of action of an endo-type enzyme.     

Effect of p58GTA on β-1,4-galactosyltransferase 1 activity and cell-cycle in human hepatocarcinoma cells

-1,4-galactosyltransferase 1 (1,4-GT 1) is the key enzyme transferring galactose to the terminal N-acetylglucosamine (GlcNAc) forming Gal14GlcNAc structure in the Golgi apparatus. In addition, it also serves as a cell adhesion molecule by recognizing and binding to terminal GlcNAc of glycoconjugates on the adjacent cell surface and matrix through a subpopulation of the enzyme distributed on the cell surface. Transient expression of the p58^GTA protein kinase, which belongs to the p34cdc2-related supergene family, could enhance 1,4-GT 1 total activity in COS cells. In this study, the p58^GTA interaction with 1,4-GT 1 was confirmed using an in vitro assay with the TNT® Coupled Reticulocyte Lysate System. An expression vector containing p58^GTA was stably transfected into 7721 cells, a human hepatocarcinoma cell line, expression was confirmed by Northern and Western blot analyses. The cells transfected with p58^GTA (p58^GTA/7721) contained 1.9 times higher total 1,4-GT 1 activity and 2.6 times higher cell-surface 1,4-GT 1 activity than the mock transfected cells (pcDNA3/7721). However, Ricinus communis agglutinin-I lectin blot analysis revealed that the enhanced 1,4-GT 1 activity did not increase the Gal14GlcNAc groups on most of the membrane proteins in p58^GTA/7721 cells. By flow cytometry analysis, it was found that the p58^GTA/7721 cells were G2/M phase arrested, compared with the pcDNA3/7721 cells. These results suggest that the p58^GTA stable transfection into human hepatocarcinoma cells could enhance the two 1,4-GT 1 subcellular pool activities independently and change its cell-cycle without modifying the -1,4-linked galactose residues on most membrane proteins.     

The menaquinol oxidase of Bacillus subtilis W23

The quinol oxidase appears to be mainly responsible for the oxidation of bacterial MKH₂ in Bacillus subtilis W23 growing with either glucose or succinate. The activity of the enzyme was maximum with dimethylnaphthoquinol, a water-soluble analogue of the bacterial menaquinol. Menadiol or duroquinol were less actively respired, and naphthoquinol was not oxidized at all. After fourtyfold purification the isolated enzyme contained 5.3 mol cytochrome aa ₃ per gram of protein and negligible amounts of cytochrome b and c. The turnover number based on cytochrome aa ₃ was about 10³ electrons · s^-1 at pH 7 and 37°C. The preparation consisted mainly of a M _r 57000 and a M _r 36000 polypeptide. The N-terminal amino acid sequence of the latter polypeptide differed from that predicted by the qoxA gene of B. subtilis strain 168 (Santana et al. 1992), in that asp-14 predicted by qoxA was missing in the M _r 36000 polypeptide.Abbreviations DMN 2,3-dimethyl-1,4-naphthoquinone - DMNH₂ 2,3-dimethyl-1,4-naphthoquinol - Duroquinol 2,3,5,6-tetramethyl-1,4-benzoquinol - MK menaquinone - MKH₂ menaquinol - NBH₂ 2,3-dimethoxy-5-methyl-6-(n-nonyl)-1,4-benzoquinol - TMPD N,N,N, N,-tetramethyl-1,4-phenylenediamine     

Hydrothermal synthesis of two novel three-dimensional cobalt(II) metal-organic frameworks based on polycarboxylate and benzotriazole

The hydrothermal reactions of 1,4-H₂BDC or 1,4-H₂CDC, HBTA, with Co(NO₃)₂ · 6H₂O in basified solvent gave rise to two coordination polymers, Co₅(μ₃-OH)₂(1,4-BDC)₃(BTA)₂ (1), [Co(1,4-CDC)_0.5(BTA)] (2) (1,4-H₂BDC = 1,4-benzenedicarboxylic acid, 1,4-H₂CDC = 1,4-cyclohexanedicarboxylic acid, HBTA = benzotriazole) and characterized by elemental analysis, IR, single-crystal X-ray diffraction and variable-temperature magnetic measurements. Complex 1 crystallizes in the triclinic system, P space group; the structure determination reveals that 1 has a scarcely reported 8-connected 3D self-penetrating structure based on pentanuclear cobalt clusters. Complex 2 is monoclinic system, P2₁/c space group, and the X-ray structural analysis shows that 2 has a 3D infinite network with (4.6⁴.8)(4².6².8²) topology. Complex 1 exhibits moderately antiferromagnetic coupling, while complex 2 indicating strong spin-orbit coupling.     

Composition and Structure of Galactomannan from the Seed of Gleditsia ferox Desf.

Galactomannan, a heteropolysaccharide with a molecular weight of 1660 kDa, was isolated from the seed of Gleditsia ferox Desf., introduced in Russia, with a yield of 18.9%. Its aqueous solutions were optically active ([]_D = +30.5°) and highly viscous ([] = 1430 ml/g). An analysis of the heteropolysaccharide using chemical, enzymatic, and chromatographic procedures showed that it consists of D-mannopyranose and D-galactopyranose residues (molar ratio, 2.54 : 1). The main chain of this galactomannan consists of 1,4--D-mannopyranose residues, 39.2% of which are substituted at C6 with single residues of -D-galactopyranose. The probability of occurrence of mannobiose units differentially substituted with galactose was determined by ¹³C-NMR data and equaled, respectively, 0.37, 0.47, and 0.16 for non-substituted Man–Man units, monosubstituted Gal(Man–Man) and (Man–Man)Gal units taken together, and for the disubstituted Gal(Man–Man)Gal units.     

Oxidation of ethanol by methanogenic bacteria

Methanogenium organophilum, a non-autotrophic methanogen able to use primary and secondary alcohols as hydrogen donors, was grown on ethanol. Per mol of methane formed, 2 mol of ethanol were oxidized to acetate. In crude extract, an NADP⁺-dependent alcohol dehydrogenase (ADH) with a pH optimum of about 10.0 catalyzed a rapid (5 mol/min·mg protein; 22°C) oxidation of ethanol to acetaldehyde; after prolonged incubation also acetate was detectable. With NAD⁺ only 2% of the activity was observed. F₄₂₀ was not reduced. The crude extract also contained F₄₂₀: NADP⁺ oxidoreductase (0.45 mol/min·mg protein) that was not active at the pH optimum of ADH. With added acetaldehyde no net reduction of various electron acceptors was measured. However, the acetaldehyde was dismutated to ethanol and acetate by the crude extract. The dismutation was stimulated by NADP⁺. These findings suggested that not only the dehydrogenation of alcohol but also of aldehyde to acid was coupled to NADP⁺ reduction. If the reaction was started with acetaldehyde, formed NADPH probably reduced excess aldehyde immediately to ethanol and in this way gave rise to the observed dismutation. Acetate thiokinase activity (0.11 mol/min·mg) but no acetate kinase or phosphotransacetylase activity was observed. It is concluded that during growth on ethanol further oxidation of acetaldehyde does not occur via acetylCoA and acetyl phosphate and hence is not associated with substrate level phosphorylation. The possibility exists that oxidation of both ethanol and acetaldehyde is catalyzed by ADH. Isolation of a Methanobacterium-like strain with ethanol showed that the ability to use primary alcohols also occurs in genera other than Methanogenium.Non-standard abbreviations ADH alcohol dehydrogenase - Ap₅ALi₃ P¹,P⁵-Di(adenosine-5-)pentaphosphate - DTE dithioerythritol (2,3-dihydroxy-1,4-dithiolbutane) - F₄₂₀ N-(N-l-lactyl--l-glutamyl)-l-glutamic acid phosphodiester of 7,8-dimethyl-8-hydroxy-5-deazariboflavin-5-phosphate - Mg. Methanogenium - OD₅₇₈ optical density at 578 nm - PIPES 1,4-piperazine-diethanesulfonic acid - TRICINE N-(2-hydroxy-1,1-bis[hydroxymethyl]methyl)-glycine - Tris 2-amino-2-hydroxy-methylpropane-1,3-diol - U unit (mol substrate/min)     

Rapid 2,2′-bicinchoninic-based xylanase assay compatible with high throughput screening**

High-throughput screening requires simple assays that give reliable quantitative results. A microplate assay was developed for reducing sugar analysis that uses a 2,2-bicinchoninic-based protein reagent. Endo-1,4--d-xylanase activity against oat spelt xylan was detected at activities of 0.002 to 0.011 IU ml^–1. The assay is linear for sugar concentrations from 0 to 86 g ml^–1 and can also be used to assay protein concentrations (0 to 143 g ml^–1) on the same plate. A variety of temperatures and pH conditions can be used and, after incubation, the assay requires only one detection reagent and one heating step.     

Glucomannan synthesis in pea epicotyls: The mannose and glucose transferases

Membrane fractions and digitonin-solubilized enzymes prepared from stem segments isolated from the third internode of etiolated pea seedlings (Pisum sativum L. cv. Alaska) catalyzed the synthesis of a -1,4-[su14C]mannan from GDP-d-[U-¹⁴C]-mannose, a mixed -1,3- and -1,4-[¹⁴C]glucan from GDP-d-[U-¹⁴C]-glucose and a -1,4-[¹⁴C]-glucomannan from both GDP-d-[U-¹⁴C]mannose and GDP-d-[U-¹⁴C]glucose. The kinetics of the membrane-bound and soluble mannan and glucan synthases were determined. The effects of ions, chelators, inhibitors of lipid-linked saccharides, polyamines, polyols, nucleotides, nucleoside-diphosphate sugars, acetyl-CoA, group-specific chemical probes, phospholipases and detergents on the membrane-bound mannan and glucan synthases were investigated. The -glucan synthase had different properties from other preparations which bring about the synthesis of -1,3-glucans (callose) and mixed -1,3- and -1,4-glucans and which use UDP-d-glucose as substrate. It also differed from xyloglucan synthase because in the presence of several concentrations of UDP-d-xylose in addition to GDP-d-glucose no xyloglucan was formed. Using either the membrane-bound or the soluble mannan synthase, GDP-d-glucose acted competitively in the presence of GDP-d-mannose to inhibit the incorporation of mannose into the polymer. This was not due to an inhibition of the transferase activity but was a result of the incorporation of glucose residues from GDP-d-glucose into a glucomannan. The kinetics and the composition of the synthesized glucomannan depended on the ratio of the concentrations of GDP-d-glucose and GDP-d-mannose that were available. Our data indicated that a single enzyme has an active centre that can use both GDP-d-mannose and GDP-d-glucose to bring about the synthesis of the heteropolysaccharide.Abbreviations CHAPS 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate - CHAPSO 3-[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate - CHD 1,2-cyclohexanedione - CDP cytidine 5-diphosphate - EGTA ethylene glycol-bis(-aminoethyl ether) N,N,N,N-tetraacetic acid - GDP guanosine 5-diphosphate - NAI N-acetyl-imidazole - NEM N-ethylmaleimide - PGO phenylglyoxal This work has been made possible by grants of M.A.F. and M.U.R.S.T. 40% of Italy. Dr. A. Zuppa wishes to thank the C.N.R. of Italy for his research scolarship.     

Isolation and culture of cotton ovule epidermal protoplasts (prefiber cells) and analysis of the regenerated wall

Culture protocols were developed and characterization of the regenerated cell walls was performed for protoplasts of cotton (Gossypium hirsutum L., L., var. Acala SJ-2) ovule epidermal cells. This work was undertaken in order to extend studies concerning nutritional effects and regulation of nucleotide sugar incorporation into -1,3- and -1,4-glucan components of cotton fiber cell walls. Protein and carbohydrate polymers and recovered from the culture medium. Analysis of a cellular fraction indicated that the majority of ¹⁴C incorporated from [¹⁴C] glucose was present in the hot-water-soluble fraction of the cells. The majority of label incorporated into cell wall material could be solubilized with acetic-nitric reagent, indicative of noncellulosic material, and characterized as -1,3-linked glucans. Only 5 to 15% of the regenerated cell wall could be characterized as -1,4-linked glucose indicative of cellulose.     

Production of N-Succinyl-l-diaminopimelic Acid by Auxotrophic Mutants of Aerobacter aerogenes and Serratia marcescens

α,ε-Diaminopimelic acid (DAP)-requiring mutants isolated from Aerobacter aerogenes ATCC 8308 and Serratia marcescens ATCC 19180 were found to accumulate N-succinyl-l-diaminopimelic acid (SDAP) which was an intermediate in the biosynthesis of lysine in Escherichia coli. SDAP was isolated from the culture broth and identified by the behavior in paper chromatography, melting point, elementary analysis, infrared spectrum, and optical rotation.

The culture conditions for SDAP production by A. aerogenes KY 7049 (DAP^?) and S. marcescens KY 8921 (DAP^?/Lys^?) were investigated. A. aerogenes KY 7049 has an absolute requirement for DAP together with a relative requirement for l-lysine. High levels of DAP (2000~4000 μg/ml) were proved to be favorable for SDAP accumulation, while if lysine along with DAP was added to the fermentation medium, optimal level of DAP for SDAP production was relatively low (about 200 μg/ml at 200 μg/ml of lysine). A variety of compounds which may conceivably affect the course of a fermentation process, i.e., carbon source, inorganic nitrogen source, amino acids, vitamines, precursors, were screened at optimal levels of lysine and DAP. Thus, the amount of SDAP accumulation reached a level of 19.9 mg/ml with the medium containing 10% glucose and 2000 μg/ml of DAP. S. marcescens KY 8921 requires either DAP or lysine for growth. Optimal level of DAP and lysine for SDAP accumulation was 50~100μg/ml.     

Carbon isotope discrimination at vegetative stage as an indicator of yield and water use efficiency of spring wheat (Triticum turgidum L. var. durum)

A field experiment was conducted to investigate if carbon isotope (¹³C) discrimination () measured at the vegetative stage of spring wheat (Triticum turgidum L. var. durum) is related with the yield and water use efficiency (WUE) at ripening. A line source sprinkler irrigation system exposed the wheat genotypes to different watering regimes, from rainfed to full irrigation and thereby increased the range in yield and WUE attainable in the four genotypes studied. The results indicated that values measured at the late stem elongation stage 60 days after planting (DAP), showed strong positive correlation with total dry matter yield (r=0.732^***), and a highly significant negative correlation with WUE (r=–0.755^***) measured at ripening 105 DAP. The data suggest that the imprints of measured at vegetative growth stage persists throughout the entire growth period, until maturity. Subject to confirmation from additional studies in other crops and locations, early measurements of may prove a useful tool for rapid and early screening of cultivars, for high yield and high WUE.     

Autophagy gets a brake: DAP1, a novel mTOR substrate,is activated to suppress the autophagic process

Autophagy, a highly regulated catabolic process, is controlled by the action of positive and negative regulators. While many of the positive mediators of autophagy have been identified, very little is known about negative regulators that might counterbalance the process. We recently identified death-associated protein 1 (DAP1) as a suppressor of autophagy and as a novel direct substrate of mammalian target of rapamycin (mTOR). We found that DAP1 is functionally silent in cells growing under rich nutrient supplies through mTOR-dependent inhibitory phosphorylation on two sites, which were mapped to Ser3 and Ser51. During amino acid starvation, mTOR activity is turned off resulting in a rapid reduction in the phosphorylation of DAP1. This caused the conversion of the protein into a suppressor of autophagy, thus providing a buffering mechanism that counterbalances the autophagic flux and prevents its overactivation under conditions of nutrient deprivation. Based on these studies we propose the “gas and brake” concept in which mTOR, the main sensor that regulates autophagy in response to amino acid deprivation, also controls the activity of a specific balancing brake to prevent the overactivation of autophagy.Key words: DAP1, mTOR, autophagy, amino acid starvation, phosphorylationIn recent years, many of the genes controlling and executing the autophagic process have been identified. Most of these genes act as positive mediators of the various steps of the process, including the ULK1 complex, which regulates the induction step, the Vps34-Beclin 1 complex that participates in the vesicle nucleation step and two ubiquitin-like pathways, the Atg12-Atg5 and the LC3-phosphatidylethanolamine (PE) conjugation steps, which play a central role in the vesicle elongation process. To date, only a few negative regulators of autophagy have been identified, including mTOR and the anti-apoptotic Bcl-2 family members. mTOR Ser/Thr kinase is a central suppressor of autophagy acting at the initiating regulatory steps of the process. Many signaling pathways act to inhibit mTOR activity, thus relieving its inhibitory effects on autophagy. The anti-apoptotic Bcl-2 and Bcl-X_L proteins, on the other hand, act at the nucleation step, by directly binding to Beclin 1''s BH3 domain, thus reducing the activation of Vps34 and subsequent autophagy. This inhibition can be relieved through dissociation of the complex, following either JNK-1 mediated phosphorylation of Bcl-2 or DAP kinase-mediated phosphorylation of the BH3 domain of Beclin 1.DAP1 is a small (∼15 kDa), ubiquitously expressed protein, rich in prolines and lacking known functional motifs. DAP1 was isolated more than a decade ago in our laboratory using a functional approach to gene cloning aimed at identifying novel mediators of IFNγ-induced cell death in mammalian cell cultures. Until recently, very little was known about the cellular and molecular functions of DAP1, mainly due to the lack of homology to other known proteins and the lack of functional motifs that could indicate a possible cellular function and studies in mammalian systems were missing.Recently, we discovered that DAP1 is another negative regulator of autophagy; yet, interestingly, its suppressive activity is selectively turned on during the autophagic process. Moreover, we found that DAP1 suppressive activity is tightly linked to the status of mTOR kinase activity. Under nutrient-rich culture conditions, DAP1 is phosphorylated by mTOR on two sites, Ser3 and Ser51, resulting in its inactivation. In response to nutrient deprivation, mTOR is inhibited and DAP1 undergoes rapid dephosphorylation. By knocking down the endogenous DAP1 and introducing either the phosphomimetic or the nonphosphorylatible DAP1 mutants, we found that the dephosphorylation leads to activation of the autophagic suppressive function of DAP1, whereas the phophorylated form is inactive. These results led to a “gas and brake” model, in which at the same time that autophagy is induced, some brakes such as DAP1 are also activated to provide a buffering mechanism that counterbalances the autophagic flux and prevents its overactivation under nutrient-deprivation conditions (Fig. 1). Notably, balancing autophagy is extremely important, since deregulated or excessive autophagy has been implicated in the pathogenesis of diverse diseases, such as certain types of neuronal degeneration and cancer and also in cellular aging.Open in a separate windowFigure 1“Gas and brake” model. During nutrient-rich conditions, active mTORC1 phosphorylates and inactivates the components of the ULK1 complex, ULK1 and Atg13, thus preventing the induction of autophagy. DAP1 is also inactivated simultaneously by mTORC1-mediated phosphorylation on Ser3 and Ser51. In addition, mTORC1 phosphorylates and activates p70S6K and 4E-BP1, which mediate the protein translation and cell growth activities of mTOR. Upon nutrient starvation, mTORC1 activity is attenuated, leading to dephosphorylation and activation of ULK1. ULK1, in turn, undergoes autophosphorylation and phosphorylates Atg13 and FIP200 resulting in ULK1 complex activation and induction of autophagy. On the other hand, activation of DAP1 by dephosphorylation, results in suppression of autophagy, thus inserting a brake into the process of autophagy. Note that the inactive proteins/complexes are faded out.The current challenge is to identify the molecular basis of the suppressive functions of DAP1 on autophagy. We have recently shown that DAP1 knockdown enhances LC3 lipidation and autophagosome accumulation both during amino acid starvation and rapamycin treatment. In addition, preliminary data indicate that the knockdown of DAP1 has no effect on mTOR complex 1 (mTORC1) activity in cells, at least during the first hours of starvation. Accordingly, DAP1 may function between the mTORC1 and the LC3 conjugation systems. The potential targets may fall into one of the multiprotein complexes functioning downstream of mTOR such as the ULK1 complex, the Vps34-Beclin 1 complex and more. Future studies will be performed to identify the molecular mechanism by which DAP1 suppresses autophagy. The lack of known functional motifs in the DAP1 protein sequence suggests that this small proline-rich protein may function as an adaptor blocking autophagy by binding to critical protein partners that still await identification.Although autophagy is primarily a protective process for the cell, it can also play a role in cell death. In response to prolonged starvation, autophagy can act either as a cell survival mechanism or be recruited as a cell death executer. In the future it would be interesting to examine whether the autophagy enhancement resulting from DAP1 knockdown contributes to increased cell death in our system or even may convert the survival properties of autophagy into death induction. This will fit the “gas and brake” model, in which autophagy, which is initially recruited as a cell survival mechanism, is converted into cell death machinery when a certain threshold is crossed due to the loss of the “brake” by the knockdown of DAP1.To date, very little is known about the putative mechanisms that restrict the intensity of the autophagic flux to maintain the continuous benefits of this process under stress. Therefore, the ability of DAP1 to counterbalance and buffer the process in a manner that is tightly linked to the status of a central player in autophagy (i.e., mTOR) is an important discovery in this field and provides a target for future drug design.     

A Confirmation of 125I-ω-Conotoxin Labeled Sites in a Crude Membrane Fraction from Chick Brain as the α1 Subunit of N-Type Calcium Channels

Omega-conotoxin GVIA (-CTX), as a selective blocker for an N-type Ca²⁺ channel, has been conveniently used in many molecular biochemical and pharmacological experiments. There has been little elucidation of ¹²⁵I--CTX binding sites (mainly the 135-kDa band) in the crude membranes from chick brain, although the characteristics of specific ¹²⁵I--CTX binding and labeling sites in chick brain membranes have been investigated in our previous research. In this work, our goal is to further identify ¹²⁵I--CTX labeling sites in chick brain membranes by using anti-B1Nt antibodies (against the N-terminal segment B1Nt of N- or P-type Ca²⁺ channel ₁-subunits). The ¹²⁵I--CTX–labeled sites in chick brain membranes could be solubilized and immunoprecipitated by using an anti B1Nt antibody. The molecular weight of the immunoprecipitated protein was determined as 135 kDa, which is inconsistent with that of the specific ¹²⁵I--CTX binding protein reported previously. Moreover, the ¹²⁵I--CTX–labeled protein could be purified by the method of preparative SDS-PAGE and recognized by anti-B1Nt antibodies in Western blotting analysis. These results indicated that anti-B1Nt antibodies could truly recognize ¹²⁵I--CTX–labeled sites as the main band of 135 kDa from chick brain membranes, and the -CTX–labeled site (mainly the 135-kDa band) should be N-type Ca²⁺ channel ₁-subunits.     

Chronic effects of cadmium on two species of mysid shrimp:Mysidopsis bahia andMysidopsis bigelowi

Two species of mysid shrimp, the sub-tropicalMysidopsis bahia and the northern temperateMysidopsis bigelowi, were exposed simultaneously to cadmium (as CdCl₂) in a continuous-flow bioassay system to determine the effect on survival and reproductive success. Temperature and salinity were maintained at 21 ± 1°C and 30,respectively. The 96-h LC50 was 110 µg ^–1 for both species. The 23-day life cycle LC50 forM. bahia was 19.5 µg ^–1 and forM. bigelowi the 27-day LC-50 was 14.8 µg ^–1. At 10 µg ^–1 a series of morphological aberrations were observed in both species at the onset of sexual maturity. Carapace malformations apparently prevented molting after the release of the initial brood and resulted in death of brooding females. As a result, although the initial reproductive rate at this concentration was successful, successive broods could not be produced. For both species in this study the no observed effect concentration was 5.1 µg ^–1; the effect concentration was 10.0 µg ^–1. Mechanisms were postulated in this study to explain the effect of cadmium on the molting process and on calcification and enzymatic reactions of osmosis.Contribution No. 257 of the U.S. Environmental Protection Agency.Contribution No. 257 of the U.S. Environmental Protection Agency.     

Na+-dependent sugar transport in a cultured epithelial cell line from pig kidney

Summary A Na⁺-dependent hexose transport system with similar characteristics to that observed in the kidney is retained in a cultured epithelial cell line from pig kidney (LLC-PK₁). The active transport of methyl-d-glucoside ( MGP), a nonmetabolizable sugar, which shares the glucose-galactose transport system in kidney cells is mediated through a Na⁺-dependent, substrate-saturable process. The kinetic analysis of the effect of Na⁺ on the uptake of MGP indicated that the Na⁺-sugar cotransport system is an affinity type system in which the binding of either sugar or Na⁺ to carrier increases the affinity for the other ligand without affecting theV _max. The sequence of selectivity for different sugars studied by the inhibition produced in the uptake of MGP is very similar to that reported in rat kidney, rabbit kidney cortex slices, and rabbit renal brush border membrane vesicles. Phlorizin, even at very low concentration, almost completely inhibits MGP uptake. Conversely, phloretin at the same low concentration stimulated the sugar accumulation by inhibition of efflux, probably at the level of the basolateral membrane. Sulfhydryl group inhibitors also blocked the MGP uptake, suggesting that these groups were required for normal functioning of the sugar carrier system. This sugar transport system is an important functional marker to study the molecular events associated with the development of polarization in epithelial cells.     

Stereospecific assignment of β-methylene protons in larger proteins using 3D15N-separated Hartmann-Hahn and13C-separated rotating frame Overhauser spectroscopy

Summary ³J _x coupling constants and complementary nuclear Overhauser data on the intraresidue C ^x H–CH distances form an essential part of the data needed to obtain stereospecific assignments of -methylene protons in proteins. In this paper we show that information regarding the magnitude of the³J _x coupling constants can be extracted from a semi-quantitative interpretation of relative peak intensities in a 3D¹⁵N-separated¹H–¹H Hartmann-Hahn¹H–¹⁵N multiple quantum coherence (HOHAHA-HMQC) spectrum. In addition, we demonstrate that reliable information on the intraresidue C ^x H–CH distances, free of systematic errors arising from spin diffusion, can be obtained from a 3D¹³C-separated¹H–¹H rotating frame Overhauser effect¹H–¹³C multiple quantum coherence (ROESY-HMQC) spectrum. The applicability of these experiments to larger proteins is illustrated with respect to interleukin-1, a protein of 153 residues and 17.4 kDa molecular weight.Abbreviations 1L-1 interleukin-1 - NOE nuclear Overhauser effect - ROE rotating frame Overhauser effect - HOHAHA homonuclear Hartmann-Hahn spectroscopy - NOESY nuclear Overhauser enhancement spectroscopy - ROESY rotating frame Overhauser spectroscopy - HMQC heteronuclear multiple quantum coherence spectroscopy     

(1,4)-β-d-Arabinoxylan arabinofuranohydrolase: a novel enzyme in the bioconversion of arabinoxylan

Summary An enzyme able to hydrolyse the terminal non-reducing -l-arabinofuranoside residues from arabinoxylans only has been found. This enzyme was unable to split arabinofuranosyl linkages in a range of other arabinofuranosyl-containing substrates. Analysis of reaction mixtures of arabinoxylan with this enzyme did not show a shift in the molecular weight distribution of the arabinoxylan, even after 24 h of incubation. Only monomeric arabinose was released. ¹H-Nuclear magnetic resonance studies of arabinoxylan treated with this enzyme, described as (1,4)--d-arabinoxylan arabinofuranohydrolase, indicated a specificity towards the single-substituted xylose in arabinoxylan.Offprint requests to: A. G. J. Voragen