Mechanism of apoptosis induced by a newly synthesized derivative of macrosphelides with a thiazole side chain

The apoptosis-inducing ability of hybrid compounds composed of macrosphelide and thiazole-containing side chain of epothilones was investigated. Among the tested series of hybrid compounds the one containing thiazole side chain at C15 (MSt-2) showed the maximum potency to induce apoptosis, while another containing thiazole side chain at C3 (MSt-6) was less potent. MSt-2 was found to induce apoptosis in human lymphoma (U937) cells in a dose- and time-dependent manner as confirmed by DNA fragmentation analysis. MSt-2 treated cells showed rapid reactive oxygen species (ROS) formation and c-Jun N-terminal kinase (JNK) activation. Furthermore, significant activation of extrinsic pathway as evident by Fas expression and caspase-8 activation was also observed. MSt-2-mediated decreased expression of Bid is an important event for cross talk between intrinsic and extrinsic signaling. N-acetyl-l-cysteine pre-treatment rescued cells from MSt-2-induced ROS formation, mitochondrial membrane potential (Δψ_m) loss, Fas expression, caspase-8 and -3 activation and DNA fragmentation. Moreover, antioxidant enzymes catalase and/or superoxide dismutase conjugated with polyethylene glycol also inhibit MSt-2-induced ROS formation, apoptosis and Δψ_m loss suggesting thereby pro-oxidant effects of MSt-2. Furthermore, JNK and pan-caspase inhibitors also protect cells from MSt-2-induced apoptosis. In addition to this, MSt-2 was found to be more potent in human colon carcinoma (HCT116) and human gastric cancer (AGS) cells while it has no effect on human normal dermal fibroblast. The important structure-activity relationship observed in the current study which makes MSt-2 more potent than MSt-6 is the position of thiazole side chain and stereochemistry of position 3 in chemical structure. In short the results of our study demonstrate that MSt-2-induced rapid ROS generation and mitochondrial dysfunction in cells trigger events responsible for mitochondria-dependent apoptosis pathway.     

Knock-out of metacaspase and/or cytochrome c results in the activation of a ROS-independent acetic acid-induced programmed cell death pathway in yeast

To gain further insight into yeast acetic acid-induced programmed cell death (AA-PCD) we analyzed the effects of the antioxidant N-acetyl-l-cysteine (NAC) on cell viability, hydrogen peroxide (H₂O₂) production, DNA fragmentation, cytochrome c (cyt c) release and caspase-like activation in wild type (wt) and metacaspase and/or cyt c-lacking cells. We found that NAC prevents AA-PCD in wt cells, by scavenging H₂O₂ and by inhibiting both cyt c release and caspase-like activation. This shows the occurrence of a reactive oxygen species (ROS)-dependent AA-PCD. Contrarily no NAC dependent change in AA-PCD of mutant cells was detectable, showing that a ROS-independent AA-PCD can also occur.     

Cardiovascular oxidative stress is reduced by an ACE inhibitor in a rat model of streptozotocin-induced diabetes

Blockade of the renin-angiotensin system (RAS) reduces cardiovascular morbidity and mortality in diabetic patients. Ang II-mediated generation of reactive oxygen species (ROS) has been suggested to be involved in several diabetic complications. We investigated whether the inhibition of Ang II production with an ACE inhibitor (ACEi) reduces oxidative stress and limits structural cardiovascular remodeling in a rat model of streptozotocin (STZ)-induced diabetes. Diabetic rats were treated for 7 weeks with an ACEi (lisinopril, 5 mg/kg/d), an antioxidant (N-acetyl-l-cysteine (NAC), 0.5 g/kg/d) and their combination. At sacrifice, ROS in the myocardium and thoracic aorta, LV myocyte number and size and aorta morphology were determined by quantitative histological methods. Superoxide and hydroxyl radical content, detected by dihydroethidium (DHE) and 8-hydroxydeoxyguanosine (8-OHdG), were 6.7 and 4.5-fold, respectively, higher in diabetic myocardium than in non-diabetic controls (p<0.001). The amount of superoxide was 5-fold higher in the thoracic aorta of diabetic rats compared to controls (p<0.001). Diabetes caused a modest increase in myocyte volume (+13%, p<0.01), a reduction of LV myocyte number (-43%, p<0.001), an accumulation of collagen around coronary arterioles (1.9-fold increase, p<0.01) and a decrease in arterial elastin/collagen ratio (-63%, p<0.001) compared to controls. Treatment with the ACEi attenuated ROS formation and prevented phenotypic changes in the heart (cardiomyocyte hypertrophy, perivascular fibrosis) and in the aorta of diabetic rats to the same extent as NAC. The absence of an additive effect, suggests a common mechanism of action, through the reduction of oxidative stress.     

Arginine antimetabolite L-canavanine induces apoptotic cell death in human Jurkat T cells via caspase-3 activation regulated by Bcl-2 or Bcl-xL

L-Canavanine, a natural L-arginine analog, is known to possess cytotoxicity to tumor cells in culture and experimental tumors in vivo. In this study, we first show that apoptotic cell death is associated with antitumor activity of L-canavanine against human acute leukemia Jurkat T cells. When Jurkat T cells were treated with 1.25-5.0mM L-canavanine for 36 h, apoptotic cell death accompanying several biochemical events such as caspase-3 activation, degradation of poly(ADP-ribose) polymerase (PARP), and apoptotic DNA fragmentation was induced in a dose-dependent manner; however, cytochrome c release from mitochondria was not detected. Under these conditions, the expression of Bcl-2 and its functional homolog Bcl-xL was markedly upregulated. The L-canavanine-induced caspase-3 activation, degradation of PARP, and apoptotic DNA fragmentation were suppressed by ectopic expression of Bcl-2 or Bcl-xL, both of which are known to play roles as anti-apoptotic regulators. These results demonstrate that the cytotoxic effect of L-canavanine on Jurkat T cells is attributable to the induced apoptosis and that L-canavanine-induced apoptosis is mediated by a cytochrome c-independent caspase-3 activation pathway that can be interrupted by Bcl-2 or Bcl-xL.     

Redox regulation of nerve growth factor-induced neuronal differentiation of PC12 cells through modulation of the nerve growth factor receptor, TrkA

We investigated the effects of the cellular redox state on nerve growth factor (NGF)-induced neuronal differentiation and its signaling pathways. Treatment of PC12 cells with buthionine sulfoximine (BSO) reduced the levels of GSH, a major cellular reductant, and enhanced NGF-induced neuronal differentiation, activation of AP-1 and the NGF receptor tyrosine kinase, TrkA. Conversely, incubation of the cells with a reductant, N-acetyl-L-cysteine (NAC), inhibited NGF-induced neuronal differentiation and AP-1 activation. Consistent with the suppression, NAC inhibited NGF-induced activation of TrkA, formation of receptor complexes comprising TrkA, Shc, Grb2, and Sos, and activation of phospholipase Cgamma and phosphatidylinositol 3-kinase. Biochemical analysis suggested that the cellular redox state regulates TrkA activity through modulation of protein tyrosine phosphatases (PTPs). Thus, cellular redox state regulates signaling pathway of NGF through PTPs, and then modulates neuronal differentiation.     

A study of l-leucine, l-phenylalanine and l-alanine transport in the perfused rat mammary gland: possible involvement of LAT1 and LAT2

The transport of l-leucine, l-phenylalanine and l-alanine by the perfused lactating rat mammary gland has been examined using a rapid, paired-tracer dilution technique. The clearances of all three amino acids by the mammary gland consisted of a rising phase followed by a rapid fall-off, respectively, reflecting influx and efflux of the radiotracers. The peak clearance of l-leucine was inhibited by BCH (65%) and d-leucine (58%) but not by l-proline. The inhibition of l-leucine clearance by BCH and d-leucine was not additive. l-leucine inhibited the peak clearance of radiolabelled l-leucine by 78%. BCH also inhibited the peak clearance of l-phenylalanine (66%) and l-alanine (33%) by the perfused mammary gland. Lactating rat mammary tissue was found to express both LAT1 and LAT2 mRNA. The results suggest that system L is situated in the basolateral aspect of the lactating rat mammary epithelium and thus probably plays a central role in neutral amino acid uptake from blood. The finding that l-alanine uptake by the gland was inhibited by BCH suggests that LAT2 may make a significant contribution to neutral amino acid uptake by the mammary epithelium.     

An efficient conversion of N-acetyl-D-glucosamine to N-acetyl-D-galactosamine and derivatives

2-Acetamido-2-deoxy-d-galactose (d-GalNAc) is an important monosaccharide widely distributed in nature. However, unlike its 4-epimer, the 2-acetamido-2-deoxy-d-glucose (d-GlcNAc), d-GalNAc is very expensive to obtain from commercial sources. Herein we report an efficient transformation that allows for the conversion of d-GlcNAc to a d-GalNAc derivative 7 in three steps and in 58.4-75% overall yields. The process was carried out on a greater than 20-g scale without the need of chromatography. The versatility of compound 7 was demonstrated by the synthesis of several useful monosaccharides and thiodisaccharides containing a d-GalNAc residue.     

l-Lactate generates hydrogen peroxide in purified rat liver mitochondria due to the putative l-lactate oxidase localized in the intermembrane space

In order to ascertain whether and how mitochondria can produce hydrogen peroxide (H₂O₂) as a result of l-lactate addition, we monitored H₂O₂ generation in rat liver mitochondria and in submitochondrial fractions free of peroxisomal and cytosolic contamination. We found that H₂O₂ is produced independently on the respiratory chain with 1:1 stoichiometry with pyruvate, due to a putative flavine-dependent l-lactate oxidase restricted to the intermembrane space. The l-lactate oxidase reaction shows a hyperbolic dependence on l-lactate concentration and is inhibited by NAD⁺ in a competitive manner, being the enzyme different from the l-lactate dehydrogenase isoenzymes as shown by their pH profiles.     

Accessibility of N-acyl-D-mannosamines to N-acetyl-D-neuraminic acid aldolase

N-Acetyl-D-neuraminic acid (NeuNAc) aldolase is an important enzyme for the metabolic engineering of cell-surface NeuNAc using chemically modified D-mannosamines. To explore the optimal substrates for this application, eight N-acyl derivatives of D-mannosamine were prepared, and their accessibility to NeuNAc aldolase was quantitatively investigated. The N-propionyl-, N-butanoyl-, N-iso-butanoyl-, N-pivaloyl-, and N-phenylacetyl-D-mannosamines proved to be as good substrates as, or even better than, the natural N-acetyl-D-mannosamine, while the N-trifluoropropionyl and benzoyl derivatives were poor. It was proposed that the electronic effects might have a significant influence on the enzymatic aldol condensation reaction of D-mannosamine derivatives, with electron-deficient acyl groups having a negative impact. The results suggest that N-propionyl-, N-butanoyl-, N-iso-butanoyl-, and N-phenylacetyl-D-mannosamines may be employed to bioengineer NeuNAc on cells.     

Growth factors sustain primordial germ cell survival, proliferation and entering into meiosis in the absence of somatic cells

It is known that mammalian primordial germ cells (PGCs), the precursors of oocytes and prospermatogonia, depend for survival and proliferation on specific growth factors and other undetermined compounds. Adhesion to neighboring somatic cells is also believed to be crucial for preventing PGC apoptosis occurring when they lose appropriate cell to cell contacts. This explains the current impossibility to maintain isolated mouse PGCs in culture for periods longer than a few hours in the absence of suitable cell feeder layers producing soluble factors and expressing surface molecules necessary for preventing PGTC apoptosis and stimulating their proliferation. In the present paper, we identified a cocktail of soluble growth factors, namely KL, LIF, BMP-4, SDF-1, bFGF and compounds (N-acetyl-L-cysteine, forskolin, retinoic acid) able to sustain the survival and self-renewal of mouse PGCs in the absence of somatic cell support. We show that under culture conditions allowing PGC adhesion to an acellular substrate, such growth factors and compounds were able to prevent the occurrence of significant levels of apoptosis in PGCs for two days, stimulate their proliferation and, when LIF was omitted from the cocktail, allow most of them to enter into and progress through meiotic prophase I. These results consent for the first time to establish culture conditions for purified mammalian PGCs in the absence of somatic cell support and should make easier the molecular dissection of the processes governing the development of such cells crucial for early gametogenesis.     

Ascorbic acid synthesis due to L-gulono-1,4-lactone oxidase expression enhances NO production in endothelial cells

As a primary antioxidant, ascorbic acid (AA) provides beneficial effects for vascular health mitigating oxidative stress and endothelial dysfunction. However, the association of intracellular AA with NO production occurring inside the endothelial cells remains unclear. In the present study, we addressed this issue by increasing intracellular AA directly through de novo synthesis. To restore AA synthesis pathway, bovine aortic endothelial cells were transfected with the plasmid vector encoding L-gulono-1,4-lactone oxidase (GULO, EC 1.1.3.8), the missing enzyme converting L-gulono-1,4-lactone (GUL) to AA. Functional expression of GULO was verified by Western blotting and in vitro enzyme activity assay. GULO expression alone did not lead to AA synthesis but the supply of GUL resulted in a marked increase of intracellular AA. When the cells were stimulated with calcium ionophore, A23187, NO production was more active in the GULO-expressing cells supplied with GUL, in comparison with the cells without GULO expression or without GUL supply, indicating that intracellular AA regulated NO production. Enhancement of NO production by intracellular AA was further verified in aortic endothelial cells obtained from eNOS knockout mice that were cotransfected with eNOS and GULO constructs. GULO-dependent AA synthesis also elevated intracellular tetrahydrobiopterin content, implicating that this essential cofactor of endothelial nitric oxide synthase (eNOS) might mediate the AA effect. The present study strongly suggests that intracellular AA plays critical roles in vascular physiology through enhancing endothelial NO production.     

Resistance to mitochondrial- and Fas-mediated apoptosis in human leukemic cells with acquired resistance to 9-beta-D-arabinofuranosylguanosine

We have previously reported that in a MOLT-4 leukemia cell line the acquired resistance to 9-beta-D-arabinofuranosylguanine (Ara-G) is due to deficiency of the activating enzymes deoxyguanosine kinase and deoxycytidine kinase [Biochem. Biophys. Res. Commun. 293 (5) (2002) 1489]. In this study we investigated whether apoptotic pathways are affected in two human T-cell lymphoblastic MOLT-4 cell lines with acquired resistance to Ara-G. In contrast to the MOLT-4 wild type cells, Ara-G resistant cells displayed no increase in caspase-3 or caspase-9 activity, DNA fragmentation, cytochrome c release or a drop in the mitochondrial membrane potential (DeltaPsi(mito)) upon Ara-G treatment. A drop in the DeltaPsi(mito) was induced in wild type cells after treatment with tributyltin, an inducer of mitochondrial permeability transition, and with carbonyl cyanide m-chlorophenylhydrazone, an uncoupling agent that reduces the DeltaPsi(mito), although not in Ara-G resistant cells. Ara-G resistant cells displayed higher levels of the anti-apoptotic protein Bcl-xL in immunoblots. A recent study indicates that Ara-G-induced apoptosis is mediated in part via the Fas pathway [Cancer Res. 43 (2047) (2002) 411]. When cells were treated with anti-Fas antibody, the wild type cell line exhibited increased caspase-3-like activity but the Ara-G resistant cells did not. Using FACS analysis and semi-quantitative PCR, 3-6-fold decreased protein levels and almost no detectable mRNA levels of Fas in the resistant cells were recorded. These data indicate that the inability to induce apoptosis via both the apoptosome pathway and the Fas pathway, due to increased levels of Bcl-xL and a lack of Fas, contributes to Ara-G resistance. This resistance to apoptosis in Ara-G resistant cells may serve to explain the overall resistance to a variety of anti-neoplastic drugs.     

Aminoguanidine and N-acetyl-cysteine supress oxidative and nitrosative stress in EAE rat brains

Abstract

Experimental autoimmune encephalomyelitis (EAE) is a well-established animal model of human multiple sclerosis (MS). We have evaluated the role of oxidative and nitrosative stress, as the causal factors in the development of EAE, responsible for the damage of cardinal cellular components, such as lipids, proteins and nucleic acids, resulting in demyelination, axonal damage, and neuronal death. EAE was induced in female Sprague-Dawley rats, 3 months old (300 ± 20 g), by immunization with myelin basic protein in combination with Complete Freund's adjuvant (CFA). The animals were divided into seven groups: control, EAE, CFA, EAE + aminoguanidine (AG), AG, EAE + N-acetyl-l-cysteine (NAC) and NAC. The animals were sacrificed 15 days after EAE induction, and the levels of nitrosative and oxidative stress were determined in 10% homogenate of the whole encephalitic mass. In EAE rats, brain NO production and MDA level were significantly increased (P < 0.001) compared to the control values, whereas AG and NAC treatment decreased both parameters in EAE rats compared to EAE group (P < 0.001). Glutathione (GSH) was reduced (P < 0.001) in EAE rats in comparison with the control and CFA groups, but increased in EAE + AG and EAE + NAC group compared to the EAE group (P < 0.01). Superoxide dismutase (SOD) activity was significantly decreased (P < 0.001) in the EAE group compared to all other experimental groups. The clinical expression of EAE was significantly decreased (P < 0.05) in the EAE groups treated with AG and NAC compared to EAE rats, during disease development.

The obtained results prove an important role of oxidative and nitrosative stress in the pathogenesis of EAE, whereas AG and NAC protective effects offer new possibilities for a modified combined approach in MS therapy.     

The Hypocrea jecorina (syn. Trichoderma reesei) lxr1 gene encodes a d-mannitol dehydrogenase and is not involved in l-arabinose catabolism

The Hypocrea jecorina LXR1 was described as the first fungal l-xylulose reductase responsible for NADPH dependent reduction of l-xylulose to xylitol in l-arabinose catabolism. Phylogenetic analysis now reveals that LXR1 forms a clade with fungal d-mannitol 2-dehydrogenases. Lxr1 and the orthologous Aspergillus nigermtdA are not induced by l-arabinose but expressed at low levels during growth on different carbon sources. Deletion of lxr1 does not affect growth on l-arabinose and l-xylulose reductase activity remains unaltered whereas d-mannitol 2-dehydrogenase activities are reduced. We conclude that LXR1 is a d-mannitol 2-dehydrogenase and that a true LXR1 is still awaiting discovery.     

H, C NMR and UV spectroscopy studies of gold(III)-tetracyanide complex with l-cysteine, glutathione, captopril, l-methionine and dl-seleno-methionine in aqueous solution

Auricyanide [Au(CN)₄]⁻ interaction with biologically important thiols, thioether and selenoether were carried out and monitored using ¹H, ¹³C NMR and UV spectroscopy. These ligands include l-cysteine, glutathione, captopril, l-methionine and dl-seleno-methionine. Thiols show very strong affinity to be oxidized into the disulfide by auricyanide, which gets reduced to aurocyanide [Au(CN)₂]⁻. l-cysteine reaction mechanism with [Au(CN)₄]⁻ was found to be dependent on reactants mole ratio. While l-methionine was completely inert toward auricyanide, dl-Se-methionine showed some reactivity with [Au(CN)₄]⁻ after raising solution pH to 12 that facilitated cyanide exchange.     

L-Glutamate in the extracellular space regulates endogenous D-aspartate homeostasis in rat pheochromocytoma MPT1 cells

In previous studies [FEBS Lett. 434 (1998) 231, Arch. Biochem. Biophys. 404 (2002) 92], we demonstrated for the first time that D-aspartate (D-Asp) is synthesized in cultured mammalian cell lines, such as pheochromocytoma 12 (PC12) and its subclone, MPT1. Our current focus is analysis of the dynamics of D-Asp homeostasis in these cells. In this communication, we show that L-glutamate (Glu) and L-Glu transporter substrates in the extracellular space regulate the homeostasis of endogenous D-Asp in MPT1 cells. D-Asp is apparently in dynamic homeostasis, whereby endogenous D-Asp is constantly released into the extracellular space by an undefined mechanism, and continuously and intensively taken up into cells by an L-Glu transporter. Under these conditions, L-Glu and its transporter substrates in the medium may competitively inhibit the uptake of D-Asp via the transporter, resulting in accumulation of the amino acid in the extracellular space. We additionally demonstrate that DL-TBOA, a well-established L-Glu transporter inhibitor, is taken up by the transporter during long time intervals, but not on a short time-scale.     

Stereoselective entry into the d-GalNAc series starting from the d-Gal one: a new access to N-acetyl-d-galactosamine and derivatives thereof

A new stereoselective preparation of N-aceyl-d-galactosamine (1b) starting from the known p-methoxyphenyl 3,4-O-isopropylidene-6-O-(1-methoxy-1-methylethyl)-β-d-galactopyranoside (10) is described using a simple strategy based on (a) epimerization at C-2 of 10 via oxidation-reduction to give the talo derivative 11, (b) amination with configurational inversion at C-2 of 11 via a S_N2-type reaction on its 2-imidazylate, (c) anomeric deprotection of the p-methoxyphenyl β-d-galactosamine glycoside 14, (d) complete deprotection. Applying the same protocol to 2,3:5,6:3′,4′-tri-O-isopropylidene-6′-O-(1-methoxy-1-methylethyl)-lactose dimethyl acetal (4), directly obtained through acetonation of lactose, the disaccharide β-d-GalNAcp-(1→4)-d-Glcp (1a) was obtained with complete stereoselectivity in good (40%) overall yield from lactose.     

Base catalysed isomerisation of aldoses of the arabino and lyxo series in the presence of aluminate

Base-catalysed isomerisation of aldoses of the arabino and lyxo series in aluminate solution has been investigated. L-Arabinose and D-galactose give L-erythro-2-pentulose (L-ribulose) and D-lyxo-2-hexulose (D-tagatose), respectively, in good yields, whereas lower reactivity is observed for 6-deoxy-D-galactose (D-fucose). From D-lyxose, D-mannose and 6-deoxy-L-mannose (L-rhamnose) are obtained mixtures of ketoses and C-2 epimeric aldoses. Small amounts of the 3-epimers of the ketoses were also formed. 6-Deoxy-L-arabino-2-hexulose (6-deoxy-L-fructose) and 6-deoxy-L-glucose (L-quinovose) were formed in low yields from 6-deoxy-L-mannose and isolated as their O-isopropylidene derivatives. Explanations of the differences in reactivity and course of the reaction have been suggested on the basis of steric effects.     

Occurrence and stress response of N-methylproline compounds in Tamarix species

A number of N-methylproline analogues have been found to accumulate in different species of Tamarix. These include N-methyl-L-proline (MP), trans-4-hydroxy-N-methyl-L-proline (M4HP) and trans-3-hydroxy-N-methyl-L-proline (M3HP). The three compounds appeared in all species but their relative and absolute levels depend upon species, ecotype and level of applied salt stress. A salt-conditioned ecotype of T. jordanis (Sodom) dramatically increased its accumulation of all proline analogues when subject to salt stress whereas a non-saline ecotype (Gilboa) showed little effect. The levels of M4HP and M3HP in T. meyeri increased with increasing salt stress whereas MP levels remained almost constant.     

Relationship between exercise intervention and NO pathway in patients with heart failure with preserved ejection fraction

Objective: Elevated levels of arginine derivatives in the NO pathway, such as asymmetric dimethylarginine (ADMA), are related to disease severity and reduced exercise capacity in heart failure (HF). We investigated the influence of exercise intervention on these parameters and on L-arginine (L-Arg) and L-homoarginine (L-hArg) in HF with preserved ejection fraction (HFpEF) patients.

Material and methods: Sixty-two patients (65?±?6 years) were included in this analysis and randomized to supervised endurance/resistance training (ET) or to usual care (UC). EDTA-plasma was analysed for NO metabolites.

Results: There were baseline associations for adjusted values of maximum workload with ADMA (r=??0.322, p?=?0.028) and L-Arg/ADMA ratio (r?=?0.331, p?=?0.015), and for the 6-min walk test (6MWT) with ADMA (r=??0.314, p?=?0.024) and L-Arg/ADMA ratio (r?=?0.346, p?=?0.015). No significant differences between UC and ET changes of NO parameters were observed at 3-month follow-up. Higher L-hArg levels were associated with a greater improvement in peak oxygen uptake (peak O₂) at follow-up: 3.4?±?2.8 vs. 1.1?±?2.9?mL/min/kg (p?=?0.005).

Conclusions: Exercise intervention did not influence NO parameters in HFpEF patients, but L-hArg was related to change in peak O₂.