Degradation of the novel herbicide ZJ0273 by Amycolatopsis sp. M3-1 isolated from soil

ZJ0273, propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy) benzylamino) benzoate, is a novel and broad-spectrum herbicide. In this study, 15 bacteria capable of utilizing ZJ0273 as the sole carbon source were isolated from soil. One of the isolates belonged to the family Amycolatopsis and was designated to Amycolatopsis sp. M3-1; at 30°C and pH 7.0, degradation rate of ZJ0273 could reach at 59.3% and 68.5% in 25 days and 60 days, respectively. Furthermore, six metabolites (M1–M6) during the degradation of ZJ0273 by Amycolatopsis sp. M3-1 were identified by a combination with multi-position ¹⁴C-labeled compounds (B-ZJ0273 and C-ZJ0273), chromatography, liquid scintillation spectrometer, and LC–MS, a novel pathway of ZJ0273 degradation by Amycolatopsis sp. M3-1 was proposed based on the identified metabolites and their biodegradation courses. ZJ0273 was initially hydrolyzed into M1 (4-(2-(4,6-dimethoxypyrimidin-2-yloxy) benzylamino) benzoic acid), then further oxidized into M3 (2-(4,6-dimethoxypyrimidin-2-yloxy) benzoic acid). M1 also could undergo a carbonylation into M2 (4-(2-(4,6-dimethoxypyrimidin-2-yloxy) benzamido) benzoic acid), and then its C–N and C–O bonds were cleaved to yield M3 (2-(4,6-dimethoxypyrimidin-2-yloxy) benzoic acid) and M4 (4,6-dimethoxypyrimidin-2-ol), respectively. Moreover, another two new metabolites, M5 (2-(4-hydroxy, 6-methoxypyrimidin-2-yloxy) benzoic acid) and M6 (2, 4-dihydroxy-pyrimidine) were found. M5 was formed through de-methyl of M3 and then hydrolyzed into M6.     

Synthesis and biological evaluation of novel 1-(4-methoxyphenethyl)-1H-benzimidazole-5-carboxylic acid derivatives and their precursors as antileukemic agents

We report here the synthesis and preliminary evaluation of novel 1-(4-methoxyphenethyl)-1H-benzimidazole-5-carboxylic acid derivatives 6(a–k) and their precursors 5(a–k) as potential chemotherapeutic agents. In each case, the structures of the compounds were determined by FTIR, ¹H NMR and mass spectroscopy. Among the synthesized molecules, methyl 1-(4-methoxyphenethyl)-2-(4-fluoro-3-nitrophenyl)-1H-benzimidazole-5-carboxylate (5a) induced maximum cell death in leukemic cells with an IC₅₀ value of 3 μM. Using FACS analysis we show that the compound 5a induces S/G2 cell cycle arrest, which was further supported by the observed down regulation of CDK2, Cyclin B1 and PCNA. The observed downregulation of proapoptotic proteins, upregulation of antiapoptotic proteins, cleavage of PARP and elevated levels of DNA strand breaks indicated the activation of apoptosis by 5a. These results suggest that 5a could be a potent anti-leukemic agent.     

Pharmacological evidence for the implication of both cyclic GMP-dependent and -independent transduction pathways within auxin-induced stomatal opening in Commelina communis (L.)

It has been previously suggested that auxin-induced stomatal opening results from at least two transduction pathways, one of which involves cyclic GMP (cGMP) as the mediator within a Ca²⁺ signalling cascade. This hypothesis was investigated further in epidermal peels of Commelina communis by comparing the effects of potential inhibitors of plant Ca²⁺-dependent enzymes on the stomatal opening responses to the auxin indolyl-3-butyric acid (IBA) and to the cGMP membrane-permeable derivative 8-bromoguanosine 3′,5′-cyclic monophosphate (8-Br-cGMP). In the 30–50 μM range, the potential plant calmodulin (CaM) antagonist N-(aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7) positively interacted with IBA but not with 8-Br-cGMP to open the stomata. The CaM antagonists W-7 (in the 10–20 μM range) and N-(aminohexyl)-1-naphthalenesulphonamide (40 μM), the potential inhibitors of plant protein kinases 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (20 and 200 μM) and K-252a (0.6 μM), and cyclosporine A and FK506, potential inhibitors of plant homologs of Ca²⁺–CaM complex (Ca²⁺/CaM)-dependent protein phosphatase 2B, prevented the IBA and 8-Br-cGMP responses by about 70% and 100%, respectively. Together, these results provide indirect pharmacological evidence that, in addition to the cGMP-dependent pathway, the auxin signal is transduced through at least one cGMP-independent pathway.     

Zinc and Zinc Chelators Modify Taurine Transport in Rat Retinal Cells

Zinc regulates Na⁺/Cl^?-dependent transporters, similar to taurine one, such as those for dopamine, serotonin and norepinephrine. This study examined the ex vivo effect of zinc (ZnSO₄), N,N,N,N-tetraquis-(2-piridilmetil)etilendiamino (TPEN) and diethylenetriaminepenta-acetic acid (DTPA), intracellular and extracellular zinc chelators, respectively, on rat retina [³H]taurine transport. Isolated cells were incubated in Locke solution with 100 nM of [³H]taurine for 25 s. Different concentrations of ZnSO₄ (0.5–200 μM) were used. Low concentrations of ZnSO₄ (30 and 40 μM) increased the transport, while higher concentrations (100, 150 and 200 μM) decreased it. Various concentrations of TPEN (1–200 μM) were added. Intermediate concentrations of TPEN (10–60 μM) significantly decreased [³H]taurine transport. The presence of TPEN, 20 μM, plus ZnSO₄ reversed the effect of TPEN alone. Several concentrations of DTPA (1–500 μM) were also investigated. Reduction of transport took place at high concentrations of the chelator (100, 250 and 500 μM). DTPA, 500 μM, plus ZnSO₄, did not modify the effect of it. These results indicate that zinc modulates taurine transport in a concentration-dependent manner, directly acting on the transporter or by forming taurine–zinc complexes in cell membranes.     

Vitamin e regulates acetylcholine receptor function of molluscan neurons

1. Intracellular recorclings were made from identified LP11, RBc4, D1 and E4 neurons in perioesophageal ganglionic ring with buccal ganglia of the mollusc Helix pomatia.2. The modulations of acetylcholine (ACh)-induced current by vitamin E in these neurons were investigated using two-microelectrode intracellular recorcling and voltage-clamp techniques.3. ACh receptors function on LP11 and RBc4 neurons was strongly regulated by intracellular calcium ions. For these ACh receptors application of 10⁻⁶ to 10⁻⁴ M vitamin E and calcium influx both induced an enhancement of the ACh-induced chloride current. Application of 10⁻⁵ to 5.10⁻⁵M arachidonic acid on the same identified LP11 and RBc4 neurons was shown to evoke a decrease of the ACh-induced chloride current.4. The elevation of calcium levels into D1 and E4 neurons induced a faint decrease of ACh-induced chloride current, but vitamin E and arachidonic acid were ineffective.5. The calmodulin inhibitor, chloropromazine (6.10^−-5M), strongly inhibited the enhancing effect of calcium influx on ACh-induced chloride current in LP11 and RBc4 neurons, but it had a weak influence on the effect of vitamin E.6. The effect of vitamin E on surface distribution of functional ACh receptors in LP11 and RBc4 neurons was found.7. Application of 10⁻⁴ to 10⁻⁶ M vitamin E (DL-α-tocopherol) triggered mechanisms, which after a 5 to 45-min period lead to appearance of functional ACh receptors on the parts of neuronal soma, which were further from the axon.8. Arachidonic acid (vitamin F) evoked a disappearance of functional ACh receptors, which were activated by vitamin E.     

Characterization of periplasmic protein BP26 epitopes of Brucella melitensis reacting with murine monoclonal and sheep antibodies

More than 35,000 new cases of human brucellosis were reported in 2010 by the Chinese Center for Disease Control and Prevention. An attenuated B. melitensis vaccine M5-90 is currently used for vaccination of sheep and goats in China. In the study, a periplasmic protein BP26 from M5-90 was characterized for its epitope reactivity with mouse monoclonal and sheep antibodies. A total of 29 monoclonal antibodies (mAbs) against recombinant BP26 (rBP26) were produced, which were tested for reactivity with a panel of BP26 peptides, three truncated rBP26 and native BP26 containing membrane protein extracts (NMP) of B. melitensis M5-90 in ELISA and Western-Blot. The linear, semi-conformational and conformational epitopes from native BP26 were identified. Two linear epitopes recognized by mAbs were revealed by 28 of 16mer overlapping peptides, which were accurately mapped as the core motif of amino acid residues ⁹³DRDLQTGGI¹⁰¹ (position 93 to 101) or residues ¹⁰⁴QPIYVYPD¹¹¹, respectively. The reactivity of linear epitope peptides, rBP26 and NMP was tested with 137 sheep sera by ELISAs, of which the two linear epitopes had 65–70% reactivity and NMP 90% consistent with the results of a combination of two standard serological tests. The results were helpful for evaluating the reactivity of BP26 antigen in M5-90.     

The response of short day plantChenopodium rubrum L. to abscisic acid and gibberellic acid treatment applied at two levels of photoperiodic induction

Abscisic acid (ABA) (5 x 10^-4M and 5 x 10^-5M) and gibberellic acid (1 x 10^-4M) was applied to the plumula ofChenopodium plants with partly (one dark period) or completely (three dark periods) fulfilled photoperiodic requirements for flowering. Morphological and cytoogical criteria were used to investigate the time-course of the differentiation of the treated shoot apices. Both substances were ineffective in increasing the mitotic activity of the shoot apex at the suboptimal level of induction. The degree of branching was temporarily stimulated by ABA and GA treatment under these conditions. Moreover, GA caused the elongation of the shoot apex. With the completely induced plants ABA hastened flowering and the rise in branching was observed in all the treatment 48 h following the application of growth substances.     

Effects of adrenergic drugs on the activity of tryptophan hydroxylase in midbrain raphe slices of the rat

The effects of α- and ß-adrenergic drugs on the activity of tryptophan hydroxylase were investigated in rat midbrain raphe slices. The tryptophan hydroxylase activity in slices was estimated by measuring the formation of 5-hydroxytryptophan (5-HTP) under inhibition of aromatic l-amino acid decarboxylase using 3-hydroxy-4-bromobenzyloxyamine (NSD 1055). Isoproterenol, a ß-adrenergic stimulant, significantly increased 5-HTP formation to 122% (P < 0.05) of control at 10⁻⁶ M and this effect was prevented by 10⁻⁶ M of propranolol, a ß-adrenergic blocker. 5-(1-Hydroxy-2-isopropylaminobutyl)-8-hydroxycarbostryril hydrochloride hemihydrate (OPC 2009), a ß-adrenergic stimulant which does not contain a catechol group, increased 5-HTP formation to 145% at 10⁻⁶ M. A-23187 at 5 × 10⁻⁷ M further enhanced the isoproterenol-stimulated 5-HTP formation to 156% of control. Dibutyryl cAMP at 10⁻² M, however, did not enhance it. 8-Bromo cAMP did not enhance the OPC 2009-stimulated 5-HTP formation, either. An α-adrenergic stimulant, clonidine, had no effect on 5-HTP formation. But an α-adrenergic blocker, yohimbine, reduced 5-HTP formation to 78% at 10⁻⁶ M. These results suggest that the activity of tryptophan hydroxylase can be controlled by a ß-adrenergic receptor coupled with adenylate cyclase via an intracellular cAMP-dependent process.     

Attachment of 99mTc to lipid vesicles containing the lipophilic chelate dipalmitoylphosphatidylethanolamine-DTTA

The binding of ^99mTc to negatively-charged and neutral unilamellar lipid vesicles was investigated in the absence and presence of the ligand diethylenetriaminepentaacetic acid (DTPA) covalently attached to the headgroup of phosphatidylethanolamine at the surface of the membrane. Even in the absence of DTPA on the membrane surface, ^99mTc reduced by Sn bound to the membrane surface but rapidly dissociated from the vesicles in the presence of plasma in vitro. When DTPA was present on the membrane surface, dissociation of ^99mTc from the vesicle surface in plasma was much reduced. The dissociation of ^99mTc from the surface of negatively-charged vesicles was less than for neutral vesicles in the absence of ligand but was markedly greater than for vesicles containing the ligand DTPA, suggesting that the binding of ^99mTc to vesicles with surface-attached DTPA could not be explained solely on the basis of the negative charge provided by the DTPA. In vitro experiments using ¹⁴C-labeled lipids as well as in vivo imaging studies indicated that dissociation of ^99mTc from the surface of the vesicle did not arise predominantly because of lipid exchange with plasma components or due to cleavage of Tc-DTPA from the vesicle surface. For vesicles with surface-attached DTPA, ^99mTc dissociation from the vesicle surface in plasma was further reduced by addition of the antioxidant ascorbate.     

Investigations of N-linked macrocycles for 111in and 90Y labeling of proteins

To simplify the synthesis of macrocyclic chelators, commercially available macrocyclic amines were condensed with halogenated acetic acid to prepare the five chelators 12N4 (DOTA), 14N4 (TETA), 15N4, 9N3 and 12N3. Only 12N4 and 9N3 showed efficient labeling of the free chelator with ¹¹¹In and ⁹⁰Y. Serum stability studies at 37 °C with In-labeled DTPA, 12N4 and 9N3 showed no loss of label over 2 days whereas, with ⁹⁰Y, only 12N4 showed stabilities comparable to DTPA. The 12N4 chelator was derivatized by attaching biotin on one N-acetate group to simulate the attachment to protein. The serum stability for both ¹¹¹In and ⁹⁰Y was identical to that of biotin derivatized DTPA and lower than that of the free chelators. Biodistribution studies in normal mice of a model protein (avidin) labeled with ⁹⁰Y via biotinylated 12N4 and biotinylated DTPA showed identical distribution at 1 day except in bone where the %ID/g for the macrocyclic-conjugated protein (3.4 ± 0.5, N = 8) was significantly (P < 0.001) lower than that of the DTPA-conjugated protein (9.4 ± 0.9, N = 7). In conclusion, macrocycles may be readily synthesized from the macrocyclic amines and several show useful stabilities with In and Y. When N-linked to a protein, the Y biodistribution was found to be superior to that of the corresponding DTPA-coupled protein.     

The effects of abscisic acid and methyl jasmonate on 1-aminocyclopropane 1-carboxylic acid conversion to ethylene in hypocotyl segments of sunflower seedlings,and their control by calcium and calmodulin

The conversion of 1-aminocyclopropane 1-carboxylic acid (ACC) to ethylene by hypocotyl segments of sunflower (Helianthus annuus L.) seedlings was inhibited by abscisic acid (ABA) and methyl jasmonate (Me-Ja), and this inhibitory effect increased with increasing concentration of both growth regulators. On the contrary, CaCl, enhanced ACC conversion to ethylene at the concentrations of 10^-4 M and 5 x 10^-4 M, however lower and higher concentrations had no significant action. CaCl, (5 x 10^-4M) seemed to magnify the inhibition of the reaction induced by ABA, whereas it reduced (5 x 10^-4M) and even abolished (10^-3M) the inhibitory action of Me-Ja. The results obtained with a Ca²⁺ chelator (EGTA), a Ca²⁺ channel blocker (nifedipine) and calmodulin antagonists (W7 and TFP), given in association with ABA or Me-Ja, suggested that calcium was involved in the inhibition of ACC conversion to ethylene by ABA and Me-Ja through an interaction with calmodulin. However, the mechanism of action of the two growth regulators seemed to be different, since all treatments which resulted in a decrease in cytosolic Ca²⁺ concentration or in calmodulin action induced a decrease in the effect of ABA and an increase in the effect of Me-Ja.Abbreviations ABA abscisic acid - ACC 1-aminocyclopropane 1-carboxylic acid - EFE ethylene for enzyme - EGTA ethylene glycol-bis-2-aminoethyl tetraacetic acid - Me-Ja methyl jasmonate - NIF nifedipine - TFP trifluoperazine dihydrochloride - W7 N-(6-aminohexyl)5-chloro-l-naphthalenesulfonamide hydrochloride     

Regeneration of plants from primary leaves of cowpea

Plants were regenerated from the in vitro cultured explants of primary leaves of cowpea (Vigna unguiculata L. Walp). Primary leaves, including the intact petiole, were excised from three-day-old seedlings and cultured on Gamborg's B5 basal medium containing 8×10^–7 M 2,4,5-trichlorophenoxyacetic acid, 1×10^–2 M L-glutamine and 1×10^–4 M adenine sulfate. Callus formed at the petiole end. Prolific shoot regeneration occurred when this callus was transferred to B5 basal medium containing 5×10^–6 M 6-benzyl-aminopurine (BAP). Regenerated shoots rooted in growth-regulator-free B5 basal medium and were established in soil.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - NAA 1-napthalene acetic acid - 2,4,5-T 2,4,5-trichloro-phenoxyacetic acid     

Two new alkaloids with carboxylic acid moieties from Portulaca oleracea and their anti-inflammatory effects

Two new alkaloids with carboxyl, identified as 8-(6,7-dihydroxy-1,2,3,4-tetrahydroisoquinolin-1-yl) octanoic acid and 5-(diethylamino)-2-(3-(diethylamino)-3-oxopropyl)-2-hydroxy-5-oxopentanoic acid named oleralkacid A and oleralkacid B, were isolated from Portulaca oleracea L. for the first time, whose structures were determined using spectroscopic methods including ¹D NMR, ²D NMR, UHPLC-ESI-QTOF/MS and circular dichroism (CD). Subsequently, the anti-inflammatory effects of compounds on lipopolysaccharide-stimulated macrophages were studied, and the results showed that the compound 1 and 2 at 5 μM and 10 μM could significantly inhibit inflammatory mediator IL-1β, respectively.     

In vitro development of plantlets from axillary buds of Acacia auriculiformis — a leguminous tree

Multiple shoots have developed from axillary buds excised from in vitro grown seedlings of Acacia auriculiformis on Gamborg's (B5) basal medium supplemented with coconut milk (5 or 10%) and benzylaminopurine (10^-6M). These shoots, if transferred individually to indole-3-acetic acid (10^-7M) or naphthaleneacetic acid (10^-6 or 10^-7M) augmented B5 medium, produced roots at their base.Abbreviations B5 Gamborg's basal medium (BM) - CM coconut milk - BA 6-benzylaminopurine - Kn kinetin - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid     

Synthesis and characterization of pyridoxine,nicotine and nicotinamide salts of dithiophosphoric acids as antibacterial agents against resistant wound infection

The pyridine-derived biomolecules are of considerable interest in developing medicinal compounds with various specific activities. Novel ammonium salts of pyridoxine, (S)-(–)-nicotine and nicotinamide with O,O-diorganyl dithiophosphoric acids (DTPA) were synthesized and characterized. The complexation of chiral monoterpenyl DTPA, including (S)-(–)-menthyl, (R)-(+)-menthyl, (1R)-endo-(+)-fenchyl, (1S,2S,3S,5R)-(+)-isopinocampheolyl derivatives, with pyridoxine and nicotine provided effective antibacterial compounds 3a,b,e,f, and 5a,b,d,f with MIC values against Gram-positive bacteria as low as 10?µM (6?µg/mL). Two selected pyridoxine and nicotine salts based on menthyl DTPA 3a and 5a were similarly active against antibiotic-resistant bacteria from burn wounds including MRSA. The compounds had enhanced amphiphilic and hemolytic properties and effectively altered surface characteristics and matrix-secreting ability of P. aeroginosa and S. aureus. MBC/MIC ratios of 3a and 5a suggested the bactericidal mode of their action. Furthermore, the compounds exhibited moderate cytotoxicity towards human skin fibroblasts (IC₅₀?=?48.6 and 57.6?µM, respectively, 72?h), encouraging their further investigation as potential antimicrobials against skin and wound infections.     

Activity of elaeochytrin A from Ferula elaeochytris on leukemia cell lines

Phytochemical investigation of the roots of Ferula elaeochytris made it possible to isolate two sesquiterpene esters, 6-anthraniloyljaeschkeanadiol (elaeochytrin A) and 4β-hydroxy-6α-(p-hydroxybenzoyloxy)dauc-9-ene (elaeochytrin B), as well as eight known compounds: 6-angeloyljaeschkeanadiol, teferidin, ferutinin, 6-(p-hydroxybenzoyl)epoxyjaeschkeanadiol, 6-(p-hydroxybenzoyl)lancerotriol, 5-caffeoylquinic acid, 1,5-dicaffeoylquinic acid and sandrosaponin IX. The cytotoxic activities of all compounds were investigated on K562R (imatinib-resistant) human chronic myeloid leukaemia and DA1-3b/M2^BCR-ABL (dasatinib-resistant) mouse leukemia cell line. Elaeochytrin A was the most active compound on both cell lines (IC₅₀ = 12.4 and 7.8 μM, respectively). It was also tested on non-resistant human promyelocytic leukemia cells (HL60, IC₅₀ = 13.1 μM) and was not toxic to normal peripheral blood mononuclear cells up to 100 μM.     

Protein Engineering of the 4-Methyl-5-Nitrocatechol Monooxygenase from Burkholderia sp. Strain DNT for Enhanced Degradation of Nitroaromatics

4-Methyl-5-nitrocatechol (4M5NC) monooxygenase (DntB) from Burkholderia sp. strain DNT catalyzes the second step of 2,4-dinitrotoluene degradation by converting 4M5NC to 2-hydroxy-5-methylquinone with the concomitant removal of the nitro group. DntB is a flavoprotein that has a very narrow substrate range. Here, error-prone PCR was used to create variant DntB M22L/L380I, which accepts the two new substrates 4-nitrophenol (4NP) and 3-methyl-4-nitrophenol (3M4NP). At 300 μM of 4NP, the initial rate of the variant expressing M22L/L380I enzyme (39 ± 6 nmol/min/mg protein) was 10-fold higher than that of the wild-type enzyme (4 ± 2 nmol/min/mg protein). The values of k_cat/K_m of the purified wild-type DntB enzyme and purified variant M22L/L380I were 40 and 450 (s⁻¹ M⁻¹), respectively, which corroborates that the variant M22L/L380I enzyme has 11-fold-higher efficiency than the wild-type enzyme for 4NP degradation. In addition, the variant M22L/L380I enzyme has fourfold-higher activity toward 3M4NP; at 300 μM, the initial nitrite release rate of M22L/L380I enzyme was 17 ± 4 nmol/min/mg protein, while that of the wild-type enzyme was 4.4 ± 0.7 nmol/min/mg protein. Saturation mutagenesis was also used to further investigate the role of the individual amino acid residues at positions M22, L380, and M22/L380 simultaneously. Mutagenesis at the individual positions M22L and L380I did not show appreciable enhancement in 4NP activity, which suggested that these two sites should be mutated together; simultaneous saturation mutagenesis led to the identification of the variant M22S/L380V, with 20% enhanced degradation of 4NP compared to the variant M22L/L380I. This is the first report of protein engineering for nitrite removal by a flavoprotein.     

Biological variation of Vanilla planifolia leaf metabolome

The metabolomic analysis of Vanilla planifolia leaves collected at different developmental stages was carried out using ¹H-nuclear magnetic resonance (NMR) spectroscopy and multivariate data analysis in order to evaluate their variation. Ontogenic changes of the metabolome were considered since leaves of different ages were collected at two different times of the day and in two different seasons. Principal component analysis (PCA) and partial least square modeling discriminate analysis (PLS-DA) of ¹H NMR data provided a clear separation according to leaf age, time of the day and season of collection. Young leaves were found to have higher levels of glucose, bis[4-(β-d-glucopyranosyloxy)-benzyl]-2-isopropyltartrate (glucoside A) and bis[4-(β-d-glucopyranosyloxy)-benzyl]-2-(2-butyl)-tartrate (glucoside B), whereas older leaves had more sucrose, acetic acid, homocitric acid and malic acid. Results obtained from PLS-DA analysis showed that leaves collected in March 2008 had higher levels of glucosides A and B as compared to those collected in August 2007. However, the relative standard deviation (RSD) exhibited by the individual values of glucosides A and B showed that those compounds vary more according to their developmental stage (50%) than to the time of day or the season in which they were collected (19%). Although morphological variations of the V. planifolia accessions were observed, no clear separation of the accessions was determined from the analysis of the NMR spectra. The results obtained in this study, show that this method based on the use of ¹H NMR spectroscopy in combination with multivariate analysis has a great potential for further applications in the study of vanilla leaf metabolome.     

Suppression of Rft1 Expression Does Not Impair the Transbilayer Movement of Man5GlcNAc2-P-P-Dolichol in Sealed Microsomes from Yeast

To further evaluate the role of Rft1 in the transbilayer movement of Man₅GlcNAc₂-P-P-dolichol (M5-DLO), a series of experiments was conducted with intact cells and sealed microsomal vesicles. First, an unexpectedly large accumulation (37-fold) of M5-DLO was observed in Rft1-depleted cells (YG1137) relative to Glc₃Man₉GlcNAc₂-P-P-Dol in wild type (SS328) cells when glycolipid levels were compared by fluorophore-assisted carbohydrate electrophoresis analysis. When sealed microsomes from wild type cells and cells depleted of Rft1 were incubated with GDP-[³H]mannose or UDP-[³H]GlcNAc in the presence of unlabeled GDP-Man, no difference was observed in the rate of synthesis of [³H]Man₉GlcNAc₂-P-P-dolichol or Man₉[³H]GlcNAc₂-P-P-dolichol, respectively. In addition, no difference was seen in the level of M5-DLO flippase activity in sealed wild type and Rft1-depleted microsomal vesicles when the activity was assessed by the transport of GlcNAc₂-P-P-Dol₁₅, a water-soluble analogue. The entry of the analogue into the lumenal compartment was confirmed by demonstrating that [³H]chitobiosyl units were transferred to endogenous peptide acceptors via the yeast oligosaccharyltransferase when sealed vesicles were incubated with [³H]GlcNAc₂-P-P-Dol₁₅ in the presence of an exogenously supplied acceptor peptide. In addition, several enzymes involved in Dol-P and lipid intermediate biosynthesis were found to be up-regulated in Rft1-depleted cells. All of these results indicate that although Rft1 may play a critical role in vivo, depletion of this protein does not impair the transbilayer movement of M5-DLO in sealed microsomal fractions prepared from disrupted cells.The lipid-linked oligosaccharyl donor, Glc₃Man₉GlcNAc₂-P-P-dolichol (mature DLO²), in protein N-glycosylation is formed in two stages in the endoplasmic reticulum (ER) (1–4). In the first stage the lipid intermediates Man-P-dolichol (Man-P-Dol), Glc-P-dolichol (Glc-P-Dol), and Man₅GlcNAc₂-P-P-dolichol (M5-DLO) are formed on the cytoplasmic leaflet of the ER with GDP-Man, UDP-Glc, and UDP-GlcNAc, serving as the glycosyl donors. The biosynthesis of the mature DLO is completed with the addition of four more mannosyl units and the formation of the triglucosyl cap in the second stage after the transbilayer movement of Man-P-Dol, Glc-P-Dol, and M5-DLO to the lumenal monolayer. Although many details about the genetics, enzymology, and regulation of these 14 glycosylation reactions are known, there is virtually nothing known about the ER proteins that are presumably required to allow the lipid-bound hydrophilic glycosyl groups to traverse the hydrophobic core of the ER bilayer.The PER5/RFT1 gene was originally identified by Walter and coworkers (5) as a gene that was up-regulated by the unfolded protein response and required for efficient protein N-glycosylation in yeast. In a related study (6), the rft1 mutation was shown to be inscrutably suppressed by p53, a soluble protein that has not been found in yeast.Helenius et al. (7) have reported evidence from metabolic labeling experiments indicating that the RFT1 gene in Saccharomyces cerevisiae encodes a protein that is involved in the flipping of M5-DLO in vivo. More recently, a point mutation in the human orthologue of the RFT1 gene has been shown to result in the accumulation of M5-DLO in fibroblasts from a patient containing an R67C amino acid substitution (8). Although these results implicate Rft1 in the transverse diffusion of M5-DLO, the topological orientation of the accumulated intermediate in the mutant cells and the precise function of the protein in the transbilayer movement of the glycolipid intermediate remain to be defined.Two reports (9, 10) have demonstrated that Rft1 is not required for the “flipping” of M5-DLO in a reconstituted proteoliposomal system, raising questions about the precise relationship between Rft1 and the M5-DLO flippase. A more recent corroborative study further characterizing the reconstituted flippase activity indicates that the in vitro assay exhibits an impressive specificity for M5-DLO (11).The current study was conducted to further explore the possible role of Rft1 in the transbilayer movement of M5-DLO in the ER. Our results establish the accumulation of chemical amounts of M5-DLO in the Rft1-depleted cells by FACE analysis, supporting the results obtained by metabolic labeling in the yeast (7) and human (8) mutant cells. However, a series of experiments conducted with sealed microsomal vesicles indicate that, although Rft1 may be required to overcome a biophysical constraint for the flipping of M5-DLO in vivo, its depletion does not hinder the flipping of M5-DLO in sealed microsomal preparations in vitro. The resemblance of these results to the loss of the requirement for the Lec35 gene (12) in the transverse diffusion and/or utilization of Man-P-Dol and Glc-P-Dol for lipid intermediate biosynthesis during disruption of intact cells is discussed.