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1.
S. Østergaard H. B. Aa. Theilgaard J. Nielsen 《Applied microbiology and biotechnology》1998,50(6):663-668
We have demonstrated that Penicillium chrysogenum possesses the l-cysteine biosynthetic enzyme O-acetyl-l-serine sulphhydrylase (EC 4.2.99.8) of the direct sulphhydrylation pathway. The finding of this enzyme, and thus the presence
of the direct sulphhydrylation pathway in P. chrysogenum, creates the potential for increasing the overall yield in penicillin production by enhancing the enzymatic activity of this
microorganism. Only O-acetyl-l-serine sulphhydrylase and O-acetyl-l-homoserine sulphhydrylase (EC 4.2.99.10) have been demonstrated to use O-acetyl-l-serine as substrate for the formation of l-cysteine. The purified␣enzyme did not catalyse the formation of l-homocysteine from O-acetyl-l-homoserine and sulphide, excluding the possibility that the purified enzyme was O-acetyl-l-homoserine sulphhydrylase with multiple substrate specificity. The purification enhanced the enzymatic specific activity
93-fold in relation to the cell-free extract. Two bands, showing exactly the same intensity, were present on a sodium dodecyl
sulphate/polyacrylamide gel, and the molecular masses of these were estimated to be 59 kDa and 68 kDa respectively. The K
m value for O-acetyl-l-serine and V
max of O-acetyl-l-serine sulphhydrylase were estimated to be 1.3 mM and 14.9 μmol/mg protein−1 h−1 respectively. The activity of the purified enzyme had a temperature optimum of approximately 45 °C, which is much higher
than the actual temperature for penicillin synthesis. Furthermore, O-acetyl-l-serine sulphhydrylase activity was to have a maximum in the range of pH 7.0–7.4.
Received: 20 March 1998 / Received revision: 27 July 1998 / Accepted: 12 August 1998 相似文献
2.
Rajdeep Chowdhury Suchandra Chowdhury Paromita Roychoudhury Chitra Mandal Keya Chaudhuri 《Apoptosis : an international journal on programmed cell death》2009,14(1):108-123
Introduction Resistance to apoptosis is a prominent feature of melanoma. Pharmacological concentration of arsenic in combination with a
widely known oxidant, menadione was explored in this study to synergistically sensitize malignant melanoma cells to apoptosis.
The molecular mechanism of apoptosis and the signaling-pathways involved were thoroughly investigated.
Materials methods and results Menadione synergized NaAsO2 to significantly increase ROS generation and facilitate the major apoptotic signaling events: alteration of mitochondrial
membrane potential, cytochrome c release and anti-apoptotic protein Bcl-2 down-regulation and subsequent activation of caspase-9 and caspase-3 followed by
poly-ADP-ribose polymerase-1 cleavage. Antioxidant N-acetyl-l-cysteine antagonized these events. Investigation of the signaling-pathway revealed significant suppression of AP-1 activity
but not NF-κB upon NaAsO2 and menadione application. An increase in p38 phosphorylation and p53 protein expression did also dictate the apoptotic response.
Suppression of p38 activation with SB203580 and inhibition of p53 expression by siRNA attenuated apoptosis. Transfection of
p53, in p53 null HCT cells augmented the apoptotic events. Moreover, the treatment also led to tumor size reduction in BALB/c mice developed by intra-dermal B16 mouse melanoma cell injection; however, it had no detectable pro-proliferative or pro-apoptotic
effect on non-tumor keratinocytes, normal fibroblasts or PBMC.
Conclusion This study thus provides an insight into innovative mechanisms of melanoma sensitization, a proper cure against which is still
elusive. Taken together, our data also provides the first evidence of arsenic activity accentuation by menadione through modulation
of specific signaling-pathways. 相似文献
3.
A simple, general scheme for the synthesis of sulfhydryl-specific alkyl alkanethiolsulfonate (RSSO2R) reagents where R is methyl, has been developed. Two new reagents, methyl aminoethanethiolsulfonate (2) and methyl benzylthiolsulfonate (3) were synthesized. These were used to modify stoichiometrically and selectively under mild conditions the sulfhydryl groups ofN-acetyl-l-cysteine ethyl ester (4),N-acetyl-l-cysteinep-nitroanilide (7), glutathione, and the A chain of bovine insulin. The corresponding -S-(-aminoethanethiol) and -S-(benzylthiol) derivatives ofl-cysteine and of the peptides were afforded. The characteristics and significance of these reactions and products are discussed. 相似文献
4.
Maheshwari A Misro MM Aggarwal A Sharma RK 《Apoptosis : an international journal on programmed cell death》2012,17(6):551-565
The present study was aimed to investigate the beneficial effects of N-acetyl-l-cysteine (NAC, 150 mg/kg bw twice/week) against testicular germ cell apoptosis in rats induced by chronic hCG administration
(100 IU/rat/day for 30 days). NAC co-treatment improved serum testosterone, prevented rise in lipid peroxidation, intracellular
H2O2 and the activities of antioxidant enzymes in germ cells. Replenishment of intracellular GSH and total antioxidant capacity
was seen. There was a marked reduction in TUNEL positive germ cells and expression of caspase-3 (p < 0.01) and PARP cleavage. Pro-apoptotic markers Fas, FasL, caspase-8 were also significantly downregulated. While Bcl-2
was fully restored, rise in Bax, caspase-9, phospho-JNK/JNK and phospho-c-Jun/c-Jun expression was significantly arrested.
Anti-apoptotic phospho-Akt/Akt and NF-κB were otherwise found upregulated. Taken together, the above findings demonstrate
that NAC intervention rescued the testicular germ cells from demise following chronic hCG treatment through regulation of
multiple signaling mechanisms of metazoan apoptosis. 相似文献
5.
Jurate Savickiene Grazina Treigyte Arunas Gineitis Ruta Navakauskiene 《In vitro cellular & developmental biology. Animal》2010,46(6):547-559
The modifications of intracellular redox balance leads to important cellular changes in many cell types. Here, a causal relationship
among redox state, granulocytic differentiation induced by all-trans retinoic acid (RA) or dibutyryl cAMP (dbcAMP) and apoptosis have been studied in the human acute promyelocytic leukaemia
HL-60 cells. The modulation of intracellular reactive oxygen species levels by d, l-buthionine-(S, R) sulfoximide (BSO), and n-acetyl-l-cysteine (NAC) caused inducer- and time-dependent or stage-specific effects on HL-60 cell growth inhibition, differentiation
and subsequent apoptosis. The presence of BSO during the commitment stage suppressed RA—but not dbcAMP-mediated differentiation,
while NAC inhibited both. BSO alone and in combination with RA or dbcAMP-induced apoptosis, which was prevented by NAC in
dbcAMP—but not in RA-treated cells. Using protein kinase C inhibitor, calphostin C, cross-talk effects between the intracellular
redox state and PKC signalling was identified by demonstrating inducer-dependent changes in cell differentiation or apoptosis,
which were associated with the changes in DNA-NF-κB binding activity. These observations suggest a critical role of redox
state in determining HL-60 cell behaviour and provide new insights into the complex effects of redox perturbations on the
intracellular signalling network via the involvement of PKC and NF-κB. 相似文献
6.
We have developed an enzymatic procedure for the enantiospecific synthesis ofN-acetyl-l-methionine with aminoacylase in an organic solvent.N-Acetyl-l-methionine was most effectively synthesized with a yield of about 90% (on the basis of thel-methionine used) when the reaction mixture, composed of 100 mm sodium acetate, 20 MMdl-methionine and aminoacylase (1000 units) immobilized on celite in 1 ml ethyl acetate saturated with 32 l 140mm sodium phosphate buffer (pH 7.0) containing 0.1 mm CoCl2, was incubated at 30°C for 24 h.N-Acetyl-l-methionine was isolated from the reaction mixture and the enantiomeric excess was 100%.d-Methionine was also isolated from the mixture with a yield of about 95% and 90% enantiomeric excess. The method is applicable to the synthesis of otherN-acetyl-l-amino acids. 相似文献
7.
Mitochondria are the main source of reactive oxygen species (ROS). The aim of this work was to verify the ROS generation in situ in HeLa cells exposed to prooxidants and antioxidants (menadione, tert-butyl hydroperoxide, antimycin A, vitamin E, N-acetyl-l-cysteine, and butylated hydroxytoluene) using the ROS-sensitive probes 6-carboxy-2,7-dichlorodihydrofluorescein diacetate di-acetomethyl ester (DCDHF) and dihydrofluorescein diacetate (DHF). Mitochondria were counterstained with the potential-sensitive probe tetramethylrhodamine methyl ester perchlorate (TMRM). Both DCDHF and DHF were able to detect the presence of ROS in mitochondria, though with distinct morphological features. DCDHF fluorescence was invariably blurred, smudged, and spread over the cytoplasm surrounding the major mitochondrial clusters. On the contrary, DHF fluorescence was sharp and delineated thin filaments which corresponded in all details to TMRM-stained mitochondria. These data suggest that DCDHF does not reach the mitochondrial matrix but is oxidized by ROS released by mitochondria in the cytosol. On the other hand, DHF enters mitochondria and reacts with ROS released in the matrix. Cytosolic (DCDHF+) ROS but not matrix (DHF+) ROS, were significantly decreased by vitamin E. N-acetyl-l-cysteine was effective in reducing DCDHF and DHF photooxidation in the medium, but was unable to reduce intracellular ROS. ROS generation was accompanied by partial mitochondrial depolarization. 相似文献
8.
David H. Baker 《Amino acids》2009,37(1):29-41
The ideal protein concept has allowed progress in defining requirements as well as the limiting order of amino acids in corn,
soybean meal, and a corn–soybean meal mixture for growth of young chicks. Recent evidence suggests that glycine (or serine)
is a key limiting amino acid in reduced protein [23% crude protein (CP) reduced to 16% CP] corn–soybean meal diets for broiler
chicks. Research with sulfur amino acids has revealed that small excesses of cysteine are growth depressing in chicks fed
methionine-deficient diets. Moreover, high ratios of cysteine:methionine impair utilization of the hydroxy analog of methionine,
but not of methionine itself. A high level of dietary l-cysteine (2.5% or higher) is lethal for young chicks, but a similar level of dl-methionine, l-cystine or N-acetyl-l-cysteine causes no mortality. A supplemental dietary level of 3.0% l-cysteine (7× requirement) causes acute metabolic acidosis that is characterized by a striking increase in plasma sulfate
and decrease in plasma bicarbonate. S-Methylmethionine, an analog of S-adenosylmethionine, has been shown to have choline-sparing activity, but it only spares methionine when diets are deficient
in choline and(or) betaine. Creatine, or its precursor guanidinoacetic acid, can spare dietary arginine in chicks. 相似文献
9.
10.
Hiroshi Kanzaki Toru Nagasawa Hideaki Yamada 《Applied microbiology and biotechnology》1986,25(2):97-100
Summary Utilizing the -replacement reaction ofStreptomyces cystathionine -lyase (EC 4.4.1.1.), an efficient production method forl-cystathionine has been established. Under optimal conditions, 50 mMl-cystathionine was synthesized from 50 mMO-succinyl-l-homoserine and 50 mMl-cysteine, added in four stages to the reaction mixture, with a substrate conversion rate of 100%. This productivity (11 gl-1 of reaction mixture) is about 3.5 times higher than that withl-homoserine andl-cysteine as substrates.Recipient of a JSPS Fellowship for Japanese Junior Scientists 相似文献
11.
Role of Oxidative Stress,Apoptosis, and Intracellular Homeostasis in Primary Cultures of Rat Proximal Tubular Cells Exposed to Cadmium 总被引:1,自引:0,他引:1
Cadmium (Cd) is a known nephrotoxic element. In this study, the primary cultures of rat proximal tubular (rPT) cells were
treated with low doses of cadmium acetate (2.5 and 5 μM) to investigate its cytotoxic mechanism. A progressive loss in cell
viability, together with a significant increase in the number of apoptotic and necrotic cells, were seen in the experiment.
Simultaneously, elevation of intracellular [Ca2+]i and reactive oxygen species (ROS) levels, significant depletion of mitochondrial membrane potential(Δ
Ψ) and cellular glutathione (GSH), intracellular acidification, and inhibition of Na+, K+-ATPase and Ca2+-ATPase activities were revealed in a dose-dependent manner during the exposure, while the cellular death and the apoptosis
could be markedly reversed by N-acetyl-l-cysteine (NAC). Also, the calcium overload and GSH depletion were significantly affected by NAC. In conclusion, exposure
of rPT cells to low-dose cadmium led to cellular death, mediated by an apoptotic and a necrotic mechanism. The apoptotic death
might be the chief mechanism, which may be mediated by oxidative stress. Also, a disorder of intracellular homeostasis induced
by oxidative stress and mitochondrial dysfunction is a trigger of apoptosis in rPT cells. 相似文献
12.
l-β-Haloalanines are physiologically active unnatural amino acids and they are useful intermediates for the synthesis of natural
and unnatural amino acids, S-linked glycopeptides, and lanthionines. In general l-β-haloalanines were prepared predominantly from l-serine via functional group transformation. Here we reported an alternative approach for the preparation of l-β-haloalanines via halogenation of protected l-cysteine esters which was obtained from l-cysteine or l-cystine, respectively. The mercapto group of protected l-cysteine esters was efficiently transformed to halo groups by triphenylphosphine/N-halosuccinimides. It has been proved to be a versatile desulfurization strategy via this functional group transformation. 相似文献
13.
Isolated membrane fractions of Chlorella fusca 211-8b obtained by french-press treatment and sonication catalyzed the oxidation of l-cysteine to l-cystine. The pH-optimum of this reaction was determined to be around 8–8.5 and a stoichiometry of 4 SH-groups oxidized for one O2 consumed was obtained. This thiol-oxidation system was specific for D-and l-cysteine; Dl-homocysteine and cysteamine were oxidized at about half the rate whereas all other thiols tested including glutathione, mercaptoethanol, mercaptopropionic acid and dithioerythritol were not oxidized by these membrane fractions. The apparent Km for l-cysteine was determined as 3.3 mmol l-1. Rates of 200 mol cysteine oxidized mg-1 chlorophyll h-1 were normally obtained. Extremely high rates of oxygen uptake were measured using l-cysteine methyl ester and l-cysteine ethyl ester. This thioloxidation system was not inhibited by mitochondrial electron-transport inhibitors such as rotenone or antimycin A, nor by the chloroplast electron-transport inhibitors 2,5-dibromothymochinone and 2,4-dinitrophenylether of iodonitrothymol. The cysteine oxidation catalyzed by C. fusca membranes was inhibited, however, by salicylhydroxamic acid, o-phenanthrolin, N,N-disalicyliden-1,3-diaminopropane 5,5-disulfonic acid, ethylenediaminetetraacetic acid, high KCN levels and by the buffers, N-[2-hydroxyl-1,1-bis(hydroxymethyl) ethyl] glycine and phosphate. This cysteine-oxidation system seems to function as a counterpart of thioredoxin-mediated light activation of enzymes, allowing reduced thiol groups to be oxidized again by O2 (dark inactivation).Abbreviation DTNB
5,5-dithio-bis(-2-nitrobenzoic acid). Ellmann reagent 相似文献
14.
Surendra Ghaskadbi 《Development genes and evolution》1987,196(1):66-68
Summary Cytochalasins A, B and H (CA, CB and CH) brought about cellular disaggregation and mortality in the developing embryos of the frog, Microhyla ornata. All three cytochalasins exhibited a dose-response relationship. CA was the most potent and its effects were significantly reduced by simultaneous additon of l-cysteine, a sulph-hydryl compound. -d-Glucosamine, a precursor of complex macromolecules important in cell adhesion, protected against the effects of CH. The effects of CB, however, were influenced neither by l-cysteine nor by -d-glucosamine. The results revealed differences in the mechanism of action of these three cytochalasins. 相似文献
15.
Summary Peroxidase-labelled lectins specific for various carbohydrate residues were used as histochemical reagents in the investigation of Hurler's syndrome. Peanut lectin was used to detect terminald-galactose, wheatgerm lectin forN-acetyl-d-glucosamine, soybean lectin forN-acetyl-d-galactosamine,Tetragonolobus lotus lectin for -l-fucose andBandeiraea S. lectin for -d-galactose. It was found that Kupffer cells in the liver and splenic reticulo-endothelial cells contain acid mucopolysaccharides which bind lectins in paraffin sections after appropriate fixation. The pattern of lectin binding suggests that such cells contain significant amounts ofd-galactose,l-fucose,N-acetyl-d-galactosamine andN-acetyl-d-glucosamine. It is likely that the last named carbohydrate is present as a polymer. Neurones contain a different carbohydrate, rich in galactose and fucose but poor inN-acetyl-d-glucosamine. This compound is resistant to lipid extraction. Hepatocytes, as a rule, do not react with lectins, most likely because of loss of the more soluble mucopolysaccharides during fixation. The results are consistent with the biochemical data of Hurler's syndrome and indicate that lectins can be a useful tool for the investigation of the cytochemistry of storage disorders. 相似文献
16.
Shouguo Shi Fengwang Ma Yonghong Li Fengjuan Feng Zengzhen Shang 《In vitro cellular & developmental biology. Plant》2012,48(1):1-6
In China, the native Lanzhou lily (Lilium davidii var. unicolor) is used as an edible lily and is an important source of ascorbic acid. However, an efficient transformation system has not
been developed for this important crop. Here, we describe the first successful genetic transformation mediated by particle
bombardment in this lily variety. We used pCAMBIA-GLDH, which contains the l
-galactono-1, 4-lactone dehydrogenase gene from apple, a hygromycin phosphotransferase gene as a selectable marker, and an intron-containing β-glucuronidase gene
as a reporter. In all, 35 transgenic lines were selected via GUS assays and PCR; from these, eight were confirmed by Southern
analyses. Transgenic plants were transferred to soil and grown under greenhouse conditions. The levels of reduced ascorbate
and total ascorbate (reduced ascorbate + oxidized ascorbate) in transgenic plants were found to be two to seven times those
in non-transgenic controls. 相似文献
17.
Sgambati E Marini M Vichi D Zappoli Thyrion GD Parretti E Mello G Gheri G 《Histochemistry and cell biology》2007,128(3):263-273
The aim of this study was to investigate the distribution of the oligosaccharides of the glycoconjugates in placentas from
pregnancies complicated by different degree of altered glycaemia. Placentas from women with physiological pregnancies (group
1), with pregnancies complicated by minor degree of glucose intolerance (group 2) and with pregnancies complicated by gestational
diabetes mellitus (GDM) treated with insulin (group 3) were collected. Ten lectins were used (ConA, WGA, PNA, SBA, DBA, LTA,
UEA I, GSL II, MAL II and SNA) in combination with chemical and enzymatic treatments. The data showed a decrease of sialic
acid linked α(2–6) to galactose/N-acetyl-d-galactosamine and an increase of N-acetyl-d-glucosamine in the placentas of the pathological groups, in particular the group 3, comparing to the group 1. A decrease
of l-fucose (LTA) and d-galactose-(β1–3)-N-acetyl-d-galactosamine, and an increase and/or appearance of l-fucose (UEA I) and N-acetyl-d-galactosamine were observed in both the pathological groups, particularly in the group 2, with respect to the group 1. In
GDM, and even in pregnancies with a simple alteration of maternal glycaemia, the changes in the distribution of oligosaccharides
could be related to alteration of the structure and functionality of the placenta. 相似文献
18.
Yu DY Zhao QL Furuta M Todoriki S Izumi K Yamakage K Matsumoto K Nomura T Kondo T 《Apoptosis : an international journal on programmed cell death》2012,17(6):636-645
The irradiation of fat-containing food forms 2-dodecylcyclobutanone (2-DCB) from palmitic acid (PA). In this study, we investigated
whether 2-DCB and PA induce apoptosis in human lymphoma U937 cells. We found that cell viability decreased by 2-DCB and apoptosis
was induced by 2-DCB and PA. 2-DCB and PA significantly enhanced the formation of intracellular reactive oxygen species (ROS).
Apoptosis induced by 2-DCB and PA was strongly prevented by an antioxidant, N-acetyl-l-cysteine. The treatment with 2-DCB and PA resulted in the loss of mitochondrial membrane potential, and Fas, caspase-8 and
caspase-3 activation. Pretreatment with a pan-caspase inhibitor (z-VAD) significantly inhibited apoptosis induced by 2-DCB
and PA. Moreover, 2-DCB and PA also induced Bax up-regulation, the reduction in Bcl-2 expression level, Bid cleavage and the
release of cytochrome c from the mitochondria to the cytosol. In addition, an increase in intracellular Ca2+ concentration ([Ca2+]i) was observed after the treatment with 2-DCB and PA. Our results indicated that intracellular ROS generation, the modulation
of the Fas-mitochondrion-caspase-dependent pathway and the increase in [Ca2+]i involved in apoptosis are induced by 2-DCB and PA in U937 cells. 相似文献
19.
Uptake of glyeine,l-cysteine,l-leucine,l-methionine,l-aspartic acid andl-lysine was investigated in resting cells ofSaccharomyces cerevisiae treated with 0.3mm actidione for blocking protein synthesis. The amino acids were taken up against substantial concentration gradients (up to
nearly 1,000∶1 for μm l-cysteine and glycine). They were present in the free form inside the cells. Their unidirectional transmembrane fluxes were
under a negative feedback control by the intracellular concentration of the amino acid involved. The amino acids tested apparently
employed more than one transport agéncies for their membrane passage, the half-saturation constants being 6.2–7.7×10−4
m for glycine, 2.5×10−4
m forl-cysteine, 6×10−5 and 4×10−4
m forl-lysine, 3×10−5 and 6×10−4
m forl-methionine, 7–18×10−5 and 1.6×10−3
m forl-aspartic acid and 6×10−5 and 2×10−3
m forl-leucine. The specificities of the transport systems are overlapping but there emerges a wide-affinity transport system for
glycine, alanine, leucine, methionine, serine, cysteine, phenylalanine, aspartic acid, asparagine, glutamic acid and tryptophan
(and possibly for other amino acids), and more specific systems for each of the following: glycine, lysine, methionine, histidine,
arginine, and aspartic and glutamic acids. Proline had the peculiar effect of stimulating the transport of all the amino acids
tested. The amino acids apparently interacted in the uptake not only by competition for the binding site but also by allotopic
inhibition (e.g.l-cysteine) and possibly stimulation (l-proline). The initial rate of uptake of amino acids and their steady-state level of distribution were characterized by identical
activation energies: 7.5 kcal/mole forl-lysine, 6.9 kcal/mole forl-aspartic acid, and 13.2 kcal/mole for glycine. 相似文献
20.
Wiriyathanawudhiwong N Ohtsu I Li ZD Mori H Takagi H 《Applied microbiology and biotechnology》2009,81(5):903-913
l-Cysteine is an important amino acid in terms of its industrial applications. We previously found marked production of l-cysteine directly from glucose in recombinant Escherichia coli cells by the combination of enhancing biosynthetic activity and weakening the degradation pathway. Further improvements in
l-cysteine production are expected to use the amino acid efflux system. Here, we identified a novel gene involved in l-cysteine export using a systematic and comprehensive collection of gene-disrupted E. coli K-12 mutants (the Keio collection). Among the 3,985 nonessential gene mutants, tolC-disrupted cells showed hypersensitivity to l-cysteine relative to wild-type cells. Gene expression analysis revealed that the tolC gene encoding the outer membrane channel is essential for l-cysteine tolerance in E. coli cells. However, l-cysteine tolerance is not mediated by TolC-dependent drug efflux systems such as AcrA and AcrB. It also appears that other
outer membrane porins including OmpA and OmpF do not participate in TolC-dependent l-cysteine tolerance. When a low-copy-number plasmid carrying the tolC gene was introduced into E. coli cells with enhanced biosynthesis, weakened degradation, and improved export of l-cysteine, the transformants exhibited more l-cysteine tolerance and production than cells carrying the vector only. We concluded that TolC plays an important role in
l-cysteine tolerance probably due to its export ability and that TolC overexpression is effective for l-cysteine production in E. coli.
Natthawut Wiriyathanawudhiwong and Iwao Ohtsu contributed equally to this work. 相似文献