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OBJECTIVE: To assess the role of Tamm-Horsfall mucoprotein (THM), obtained from normal subjects (nTHM) and stone-formers (sfTHM), on crystallization of calcium oxalate (CaOx) in whole urine, and to assess the effects of N-acetylcysteine (AC) in preventing renal stone formation. MATERIALS AND METHODS: A modified rapid evaporation method was used to test the effects of THM and AC on crystallization of CaOx in vitro. Animal experiments involving 30 rats and a clinical study involving 17 patients with idiopathic CaOx urolithiasis were also performed to identify further the role of AC in preventing CaOx stone formation in vivo. RESULTS: The results showed that the volume of CaOx crystals was greatly reduced, by ultrafiltration, to 39.1% of untreated urine, but increased to 81.4% by adding nTHM (35 mg/l). sfTHM was a more potent promoter. AC was found to inhibit the transformation of THM and lower the crystallization of CaOx both in vitro and in vivo. CONCLUSION: We believe that the transformation of THM is an important step in the formation of urolithiasis. AC might be a new way to prevent renal stone formation and recurrence.Fan J, Shen SJ. The role of Tamm-Horsfall mucoprotein in calcium oxalate crystallization. N-acetylcysteine�a new therapy for calcium oxalate urolithiasis. Br J Urol 74:288-293; 1994.
IL-2 therapy can induce marked oxidative stress via reactive oxygen and nitrogen intermediates. Glutathione, the major intracellular reductant, may become rate limiting to cytotoxic lymphocyte activation and proliferation under these circumstances. N-Acetyl cysteine (NAc-cys) was used to increase intracellular glutathione levels during lymphokine-activated killer (LAK) cell activation by IL-2. Incubation of splenocytes with NAc-cys (0.6 to 1.0 mM) resulted in significant changes in intracellular reduced and total glutathione (92% and 58% increase, respectively) at 96 h. These levels correlated with markedly enhanced cell proliferation (threefold) and cytolytic effector cell generation (> fivefold increase in LU/10(6) cells) induced by the combination of NAc-cys with IL-2. IL-2 exposure by itself unexpectedly increased intracellular reduced glutathione by 43%. IL-2 and NAc-cys were synergistic in increasing glutathione levels (reduced glutathione: 292% increase; total: 251% increase). Inhibition of glutathione synthesis, using L-buthionine-(S,R)-sulfoximine reversed the effects of NAc-cys on intracellular glutathione, as well as cellular proliferation and cytotoxicity. This experiment established that the effects of NAc-cys required de novo glutathione synthesis. In conjunction with IL-2/LAK treatment, oral NAc-cys administration (260 to 900 mg/kg/day for 7 days) significantly decreased tumor progression in a refractory s.c. tumor model. A small fraction of mice (11 to 17%) had complete tumor regressions. NAc-cys may be useful as an adjunct to increase the antitumor activity of IL-2/LAK therapy. Yim CY, Hibbs JB Jr, McGregor JR, et al. Use of N-acetyl cysteine to increase intracellular glutathione during the induction of antitumor responses by IL-2. J Immunol 152:5796-5805; 1994.
HIV-infected individuals and SIV-infected rhesus macaques have, on the average, decreased plasma cysteine and cystine concentrations and decreased intracellular glutathione levels. We show that the cysteine supply and the intracellular glutathione levels have a strong influence on the T cell system. A study of healthy human subjects revealed that persons with intracellular glutathione levels of 20-30 nmol/mg protein had significantly higher numbers of CD4+ T cells than persons with either lower or higher glutathione levels. Persons who moved during a 4-week observation period from the optimal to the suboptimal range (10-20 nmol/mg) experienced, on the average, a 30% decrease in CD4+ T cell numbers. This decrease was prevented by treatment with N-acetyl-cysteine (NAC). NAC caused this relative increase of CD4+ T cell numbers in spite of decreasing glutathione levels and not by increasing the glutathione level. Our studies suggest that the immune system may be exquisitely sensitive not only against a cysteine and glutathione deficiency but also against an excess of cysteine.Kinscherf R; Fischbach T; Mihm S; et al. Effect of glutathione depletion and oral N-acetyl-cysteine treatment on CD4+ and CD8+ cells. FASEB J 8:448-451; 1994.
The action of N-acetylcysteine on hepatic glycogen deposition was investigated. Emphasis was also given to the protective action of this compound against hepatic glycogen depletion by paracetamol [acetominophen]. Rats fasted for 24 hours were injected intraperitoneally with (1) vehicle, (2) paracetamol (500 mg/kg), (3) N-acetylcysteine (1200 mg/kg) and (4) paracetamol plus N-acetylcysteine. The rats were refed immediately after the drug injections. Paracetamol inhibited glycogen deposition in the 12 hours following injection. The plasma levels of paracetamol were in the range that inhibits energy metabolism in isolated mitochondria and in the isolated perfused liver. N-Acetylcysteine increased the rate of glycogen deposition either in the presence or in the absence of paracetamol. The latter effect may be responsible, partly at least, for the protective action of N-acetylcysteine against glycogen depletion caused by toxic doses of paracetamol.Itinose AM; Doi-Sakuno ML; Bracht A. N-acetylcysteine stimulates hepatic glycogen deposition in the rat. Res Commun Chem Pathol Pharmacol 83:87-92; 1994.
Glutathione is a tripeptide that contains an important thiol (sulfhydryl) group within the central cysteine amino acid. Glutathione is involved in numerous vital processes where the reducing potential of the thiol is used. Several lung disorders are believed to be characterized by an increase in alveolar oxidant burden, potentially depleting alveolar and lung glutathione. Low glutathione has been linked to abnormalities in the lung surfactant system and the interaction between glutathione and antiproteases in the epithelial lining fluid of patients. Normal levels of intracellular glutathione may exert a critical negative control on the elaboration of proinflammatory cytokines. The increase of intracellular reactive oxygen species is believed to correlate with the activation of NF-kappa B, a strongly implicate free radical injury in the genesis and maintenance of several lung disorders in humans. This information is substantial and will help the development of clinical studies examining a variety of inflammatory lung disorders.Morris PE;Bernard GR. Significance of glutathione in lung disease and implications for therapy. Am J Med Sci 307:119-127;1994
Chronic bronchitis is common among smokers, often together with recurrent infectious exacerbations. Streptococcus pneumoniae and Haemophilus influenzae are the pathogens traditionally considered most important. N-acetylcysteine (NAC) treatment has been shown to reduce the number of infectious exacerbations in patients with chronic bronchitis. The mechanism behind this is unknown. We attempted to characterize the intrabronchial bacterial flora in patients with chronic bronchitis in an infection-free interval, and to determine whether pharmacological and immunological factors effected the bacterial occurrence. Twenty two smokers with non-obstructive chronic bronchitis, 19 smokers with chronic bronchitis and chronic obstructive pulmonary disease (COPD) and 14 healthy nonsmokers underwent bronchoscopy. To obtain uncontaminated intrabronchial samples, a protected specimen brush was used. Quantitative bacterial cultures and virus isolations were performed. Significantly positive bacterial cultures (> 1,000 colony-forming units (cfu).ml-1) were found only in the patients. S. pneumoniae and H. influenzae were found in five patients, and only in the patients without NAC treatment. The most common bacterium was alpha-haemolytic streptococcus. Negative cultures were more common in the healthy controls. Of the various factors examined, only NAC medication had an influence on bacterial numbers. Significantly fewer patients with NAC medication had positive cultures (3 out of 16) than in the group of patients without NAC therapy (15 out of 21). Our results confirm that chronic bronchitis in smokers leads to increased intrabronchial bacterial colonization. We could also confirm that 1,000 cfu.ml-1 is an adequate cut-off level for significant bacterial growth when using the protected specimen brush. NAC medication was associated with low bacterial numbers.Riise GC, Larsson S, Larsson P, et al. The intrabronchial microbial flora in chronic bronchitis patients: a target for N-acetylcysteine therapy? Eur Respir J 7:94-101; 1994.
The effect of in vivo and in vitro N-acetylcysteine (NAC) treatment on destructive activity of macrophages against Candida from COPD patients has been evaluated. Patients received NAC (600 mg) or placebo orally 3 times a day for 15 days and bronchoalveolar lavage (BAL) fluid and peripheral blood were collected before and at the conclusion of treatment. In our system, NAC treatment was not able to modulate antifungal activity of alveolar macrophages, peripheral blood monocytes (PBM), and polymorphonuclear leukocytes. On the contrary, in vitro NAC treatment at appropriate doses (10 micrograms/ml) significantly enhanced antifungal activity of PBM from COPD patients. This phenomenon is mediated by augmented phagocytic activity and phagosome-lysosome fusion. The lack of correlation between in vivo and in vitro studies could be ascribed to differences in the intracellular concentration of the drug that in vivo does not reach levels capable of inducing macrophage activation. We speculate that in COPD patients who undergo long-term NAC treatment, appropriate schedules and doses of the drug could augment resistance against microbial infections which are often life-threatening in these patients.Vecchiarelli A, Dottorini M, Pietrella D, et al. Macrophage activation by N-acetyl-cysteine in COPD patients. Chest 105:806-811; 1994.
Abstract: A 32-year-old man was brought to the emergency department 5 1/2 hours after ingesting a potentially lethal dose (900 mg) of sodium arsenate ant poison in a suicide attempt. The patient deteriorated progressively for 27 hours. After intramuscular dimercaprol and supportive measures failed to improve his condition he was given N-acetylcysteine intravenously. The patient showed remarkable clinical improvement during the following 24 hours and was discharged from the hospital several days later.Martin D, Willis S, Cline D. N-Acetylcysteine In The Treatment Of Human Arsenic Poisoning. J Am Board Fam Pract 3:293-296;1990
Hepatitis C virus (HCV) is an RNA virus that replicates in both the liver and lymphoid cells. Interferon-alpha (IFN-alpha) is a useful treatment of chronic hepatitis C (CHC) although resistance to this drug occurs frequently. The mechanisms underlying resistance to IFN remain unknown. In this work, we have measured the levels of glutathione in plasma and peripheral lymphoid cells from 15 healthy controls and 24 CHC patients, 10 of whom were without treatment and 14 showed high serum alanine aminotransferase (ALT) values despite therapy with lymphoblastoid IFN for more than 4 months. In all patients, glutathione levels in plasma and in mononuclear cells were depressed in comparison to controls. In IFN-unresponsive patients, the addition of 600 mg tid of oral N-acetyl cysteine (NAC), a glutathione precursor, resulted in a steady decrease of ALT values in all patients, with complete normalization in 41% of cases after 5-6 months of combined therapy. Administration of NAC alone for 1 month was without effect in the 10 patients that were not receiving IFN. Supplementation of IFN with NAC induced a near normalization of intralymphocytic glutathione, but plasma levels were only moderately increased. HCV replication was markedly inhibited in lymphocytes and viremia was cleared in one of the 8 patients tested. In conclusion, NAC enhances the response to IFN in CHC. Controlled studies are needed to ascertain whether antioxidant therapy might act in synergy with IFN in chronic viral hepatitis.Beloqui O; Prieto J; Suarez M; et al. N-acetyl cysteine enhances the response to interferon-alpha in chronic hepatitis C: a pilot study. J Interferon Res 13:279-282; 1993.
The most commonly used chemopreventive agents in the prevention of oral leukoplakia, head and neck cancer, and lung cancer are beta-carotene, vitamin A, and other retinoids. One of the few chemopreventive agents not in this group and presently being used in a clinical trial is N-acetyl-l-cysteine (NAC). NAC, an antioxidant, is used in EUROSCAN, a European Organization of Research and Treatment of Cancer (EORTC) chemoprevention study in curatively treated patients with oral, laryngeal, or lung cancer. The rationale for choosing NAC is based on a variety of experimental data showing its ability to exert protective effects, including extracellular inhibition of mutagenic agents from exogenous and endogenous sources, inhibition of genotoxicity of reactive oxygen species, modulation of metabolism coordinated with blocking of reactive metabolites, protection of DNA and nuclear enzymes, and prevention of the formation of carcinogen-DNA adducts. NAC has also demonstrated an effect on mutagen-induced chromosomal sensitivity assays, and on anticarcinogenicity in experimental animal models. In addition, preliminary data from EUROSCAN show good compliance in treated patients and a low frequency of side effects. De Vries N, De Flora S. N-acetyl-l-cysteine. J Cell Biochem Suppl 17F:270-277; 1993.
The observations that people infected with HIV suffer not only from an inflammatory stress but also from depleted glutathione levels have led to a general hypothesis that these two are causally related, and that treatment of AIDS should include thiol-replenishment therapy. In particular, inflammatory stimulations are dependent on intracellular thiol levels, as they are potentiated at low glutathione levels (oxidative stress) and inhibited at high glutathione levels. Inflammatory stress may itself lead to decreased levels of glutathione. HIV has taken advantage of inflammatory signals to regulate its own replication; thus, the HIV infection is exacerbated by low levels of glutathione. We have shown that N-acetylcysteine can inhibit inflammatory stimulations, including that of HIV replication. Since N-acetylcysteine can replenish depleted glutathione levels in vivo, we suggest that it be used as an adjunct in the treatment of AIDS. Roederer M, Staal FJ, Ela SW, Herzenberg LA, Herzenberg LA. N-acetylcysteine: potential for AIDS therapy. Pharmacology 46:121-129; 1993.
A series of clinical studies and laboratory investigations suggests that the acquired immunodeficiency syndrome (AIDS) may be the consequence of a virus-induced cysteine deficiency. HIV-infected persons at all stages of the disease were found to have decreased plasma cystine and cysteine concentrations and decreased intracellular glutathione levels. In rhesus macaques, cysteine levels decrease already within 1-2 weeks after infection with the closely related virus SIV(mac). HIV-infected persons and SIV-infected rhesus macaques have also, on the average, substantially increased plasma glutamate levels. Increased glutamate levels aggravate the cysteine deficiency by inhibiting the membrane transport of cystine. Even moderately elevated extracellular glutamate levels as they occur in HIV-infected persons cause a substantial decrease of intracellular cysteine levels. Clinical studies revealed that individual cystine and glutamate levels are correlated with the individual lymphocyte reactivity and T4+ cell counts but not T8+ cell counts. This phenomenon was demonstrated not only in HIV-infected persons but also in healthy human individuals. The cellular cysteine supply affects amongst others the intracellular glutathione level and IL-2-dependent proliferation of T cells and (inversely) also the activation of the transcription factor NF-kappaB. The cysteine deficiency of HIV-infected persons is, therefore, possibly responsible not only for the cellular dysfunction but also for the overexpression of tumor necrosis factor-alpha (TNF-alpha), interleukin-2 receptor alpha-chain, and beta2-microglobulin. All the corresponding genes are associated with kappaB-like enhancer sequences. We have suggested that N-acetyl-cysteine may be considered for the replenishment of cysteine and glutathione levels in HIV-infected patients, since N-acetyl-cysteine is a well-established drug in clinical medicine with well-documented pharmacokinetics and safety.Droge W. Cysteine and glutathione deficiency in AIDS patients: a rationale for the treatment with N-acetyl-cysteine. Pharmacology 46:61-65; 1993.
The clinical effect of N-acetylcysteine (NAC) controlled-release tablets 300 mg b.i.d., and placebo, in chronic bronchitis was investigated. The study was performed as a double-blind six month comparison between active drug and placebo in two parellel groups, with statistical evaulation after four and six months The patients were chosen from nine centres. One hundred and sixteen out-patients were included and ninety one of them completed the six month study. The acetylcysteine-treated group had a significantly reduced number of sick-leave days caused by exacerbations of chronic bronchitis after the four winter months December-March compared with the control group (NAC 173, placebo 456). The number of exacerbation days was also very much reduced, however not significantly (NAC 204, placebo 399). At the end of the six month trial, including also two spring months the absolute numbers of sick-leave days and exacerbation days were still fewer in the acetylcysteine-treated group, (NAC 260, placebo 739) and (NAC 378, placebo 557) respectively. This study demonstrated a significant reduction in sick-leave days after four months of NAC-treatment. A constant tendency to reduction in the number of exacerbations and exacerbation days was also registered after four and six months. The differences in these parameters were, however, not statistically significant. This was probably due to the small number of patients participating.Rasmussen JB, Glennow C. Reduction in days of illness after long-term treatment with N-acetylcysteine controlled release tablets in patients with chronic bronchitis. Eur Respir J. 1988, 1, 351-355.
Thirty-six patients, 22 males and 14 females, aged about 7 years, suffering from serous otitis media (glue ear), mostly bilateral and characterized by abundant viscous discharge, underwent surgery consisting of myringotomy, aspiration and insertion of Shepard�s transtympanic drain. After surgery 18 of these patients received 200 mg oral acelylcysteine (NAC) b.i.d. for 30 days, while the remaining patients received no treatment and acted as controls. Patients were randomly assigned to one of the two groups. Audiometric tests at the various frequencies most affected by the disease, i.e. those between 250 and 3000 Hz, were performed in all patients before surgery and after 30 days of treatment. Statistical comparison between mean bearing threshold values recorded in patients of the treated and control groups before and after oral NAC treatment showed that: 1. Hearing had markedly and very significantly improved in both groups after surgery; 2. Hearing had improved more significantly in the treated group than in the control group; 3. Hearing improvement in the treated group was more marked at the low and medium frequencies particularly affected by the disease. These findings confirmed the pharmacological effect of oral NAC in the middle ear, and showed that it enhanced recovery of hearing. Follow-up took place in all patients in the treated and control groups one year after surgery. Drains had remained in situ for periods ranging from 3 to 12 months, and all had been removed or spontaneously expelled when the follow-up control was made. Otomicroscopic examination disclosed some pathological changes in both groups. However, the incidence of these changes was not significant in either group. Audiometric tests showed, with respect to both groups and to all the frequencies tested, a slight reduction in the hearing gain recorded at the previous test performed one month after the insertion of the transtympanic drains. This loss was however less pronounced in the treated group. In conclusion, oral NAC may be useful in enhanceing the functional effects of surgery in serous otitis media.Balli R. Controlled trial on the use of oral acetylcysteine in the treatment of glue-ear following drainage. Eur J Respir Dis 61, Suppl 111;158; 1980.
Rats were exposed to mercury vapors (30 mg/m3) for either I or 2 h. Histological lesions like alveolar oedema, hyaline membranes and sometimes fibrosis were observed. The lesions were more significant after 2 h of exposure, with about 50% of the animals dying within 2 weeks. The mercury level and the superoxide dismutase activity in the blood and the lungs demonstrated differences according to the time of exposure. In the animals exposed for 2 h to mercury vapors, N-acetylcysteine treatment increased survival time and the percentage of living animals. The lung superoxide dismutase was lower than in the non-treated animals indicating an antioxidant effect. Mercury levels were decreased in blood and lung, suggesting some chelating effect of N-acetylcysteine. The exact mechanism of its action must be further elucidated.Livardjani F, Lediga M, Koppa P, et al. Lung and blood superoxide dismutase activity in mercury vapor exposed rats: effect of N-acetylcysteine treatment. Toxicology 66:289-295; 1991.
Record 1 from database: MEDLINE
Hydroxyl radical footprints and half-site arrangements of binding sites for the CysB transcriptional activator of Salmonella typhimurium.
Hryniewicz MM; Kredich NM
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Bacteriol, 1995 May, 177:9, 2343-53
CysB is a transcriptional activator for the cysteine regulon and negatively autoregulates its own gene, cysB. Transcription activation also requires an inducer, N-acetyl-L-serine. CysB is known to bind to activation sites just upstream of the -35 regions of the positively regulated cysJIH, cysK, and cysP promoters and to a repressor site centered at about +1 in the cysB promoter. Additional accessory sites have been found in positively regulated promoters. The hydroxyl radical footprinting experiments reported here indicate that the activation sites CBS-J1, CBS-K1, and CBS-P1 in the cysJIH, cysK, and cysP promoters are composed of two convergently oriented 19-bp half-sites separated by 1 or 2 bp. N-Acetyl-L-serine stimulates binding to these sites as well as to the accessory sites CBS-J2 and CBS-P2, both of which share a similar topology with activation sites. A second topology is found in the accessory site CBS-K2 and the repressor site CBS-B, which contain divergently oriented 19-bp half-sites separated by one or two helical turns. N-Acetyl-L-serine inhibits binding to these two sites. A third topology is present in the cysK and cysP promoters, where an additional half-site is oriented toward the activation site and separated from it by one helical turn. Here, CysB binds to all three half-sites, bending the DNA, and N-acetyl-L-serine decreases the extent of bending. The marked dissimilarities of these half-site arrangements and of their responses to N-acetyl-L-serine suggest that CysB, a homotetramer, binds to them with different combinations of subunits.
LA=ENG
95247666
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Bacterial Proteins|*ME; DNA-Binding Proteins|*ME; DNA, Bacterial|DE/*GE; Promoter Regions (Genetics)|DE/*GE; Salmonella typhimurium|*GE; Transcription Factors|*ME
Base Sequence; Binding Sites|DE/GE; Cysteine|BI; Gene Expression Regulation, Bacterial; Hydroxyl Radical|PD; Models, Genetic; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation|DE; Protein Binding|GE; Protein Conformation; Serine|AA/PD; Support, U.S. Gov't, P.H.S.; Transcription, Genetic
JOURNAL ARTICLE
0021-9193
UNITED STATES
0 (Bacterial Proteins); 0 (CysB protein); 0 (DNA-Binding Proteins); 0 (DNA, Bacterial); 0 (Transcription Factors); 16354-58-8 (N-acetylserine); 3352-57-6 (Hydroxyl Radical); 4371-52-2 (Cysteine); 56-45-1 (Serine)
Record 1 from database: MEDLINE
Stoichiometry of binding of CysB to the cysJIH, cysK, and cysP promoter regions of Salmonella typhimurium.
Hryniewicz MM; Kredich NM
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
J Bacteriol, 1994 Jun, 176:12, 3673-82
CysB is a member of the LysR family of transcriptional activators and regulates genes of the cysteine regulon in Salmonella typhimurium and Escherichia coli. CysB binds to specific sites just upstream of the -35 regions of the cysJIH, cysK, and cysP promoters, where, in the presence of N-acetyl-L-serine, it stimulates transcription initiation. The cysK and cysP promoters contain additional binding sites, and we have proposed that CysB bends these promoters by binding to adjacent sites. N-Acetyl-L-serine is thought to decrease the magnitude of such bending. Since stoichiometric data bearing on this model have been lacking, we analyzed complexes in gel mobility shift experiments with 35S-labeled CysB and 32P-labeled promoter fragments. CysB was found to bind as a tetramer, and N-acetyl-L-serine increased the electrophoretic mobilities of one-protein complexes of the multibinding site cysK and cysP promoters without changing their stoichiometry, indicating that a single CysB tetramer can bend these promoters and that N-acetyl-L-serine diminishes such bending. Bend angles for both promoters were calculated to be 100 and 50 degrees in the absence and presence of N-acetyl-L-serine. N-Acetyl-L-serine affected neither the stoichiometry nor the electrophoretic mobility of cysJIH promoter complexes, which are not known to contain bent DNA. DNA bending may be a mechanism for sequestering CysB at certain promoter sites by increasing their affinity for this protein in the absence of N-acetyl-L-serine.
LA=ENG
94266720
Bacterial Proteins|GE/*ME/PD; DNA-Binding Proteins|*ME; Genes, Bacterial|*GE; Promoter Regions (Genetics)|*GE; Salmonella typhimurium|*GE/ME
Comparative Study; Cysteine|BI; DNA, Bacterial|CH/ME; Models, Genetic; Nucleic Acid Conformation|DE; Protein Binding; Protein Conformation; Regulon|GE; Serine|AA/PD; Support, U.S. Gov't, P.H.S.; Transcription Factors|GE; Transcription, Genetic
JOURNAL ARTICLE
0021-9193
UNITED STATES
0 (Bacterial Proteins); 0 (CysB protein); 0 (DNA-Binding Proteins); 0 (DNA, Bacterial); 0 (Transcription Factors); 16354-58-8 (N-acetylserine); 4371-52-2 (Cysteine); 56-45-1 (Serine); 87609-37-8 (LysR protein)
Record 2 from database: MEDLINE
Hydroxyl radical footprints and half-site arrangements of binding sites for the CysB transcriptional activator of Salmonella typhimurium.
Hryniewicz MM; Kredich NM
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Bacteriol, 1995 May, 177:9, 2343-53
CysB is a transcriptional activator for the cysteine regulon and negatively autoregulates its own gene, cysB. Transcription activation also requires an inducer, N-acetyl-L-serine. CysB is known to bind to activation sites just upstream of the -35 regions of the positively regulated cysJIH, cysK, and cysP promoters and to a repressor site centered at about +1 in the cysB promoter. Additional accessory sites have been found in positively regulated promoters. The hydroxyl radical footprinting experiments reported here indicate that the activation sites CBS-J1, CBS-K1, and CBS-P1 in the cysJIH, cysK, and cysP promoters are composed of two convergently oriented 19-bp half-sites separated by 1 or 2 bp. N-Acetyl-L-serine stimulates binding to these sites as well as to the accessory sites CBS-J2 and CBS-P2, both of which share a similar topology with activation sites. A second topology is found in the accessory site CBS-K2 and the repressor site CBS-B, which contain divergently oriented 19-bp half-sites separated by one or two helical turns. N-Acetyl-L-serine inhibits binding to these two sites. A third topology is present in the cysK and cysP promoters, where an additional half-site is oriented toward the activation site and separated from it by one helical turn. Here, CysB binds to all three half-sites, bending the DNA, and N-acetyl-L-serine decreases the extent of bending. The marked dissimilarities of these half-site arrangements and of their responses to N-acetyl-L-serine suggest that CysB, a homotetramer, binds to them with different combinations of subunits.
LA=ENG
95247666
Bacterial Proteins|*ME; DNA-Binding Proteins|*ME; DNA, Bacterial|DE/*GE; Promoter Regions (Genetics)|DE/*GE; Salmonella typhimurium|*GE; Transcription Factors|*ME
Base Sequence; Binding Sites|DE/GE; Cysteine|BI; Gene Expression Regulation, Bacterial; Hydroxyl Radical|PD; Models, Genetic; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation|DE; Protein Binding|GE; Protein Conformation; Serine|AA/PD; Support, U.S. Gov't, P.H.S.; Transcription, Genetic
JOURNAL ARTICLE
0021-9193
UNITED STATES
0 (Bacterial Proteins); 0 (CysB protein); 0 (DNA-Binding Proteins); 0 (DNA, Bacterial); 0 (Transcription Factors); 16354-58-8 (N-acetylserine); 3352-57-6 (Hydroxyl Radical); 4371-52-2 (Cysteine); 56-45-1 (Serine)
Record 3 from database: MEDLINE
The protease inhibitor, N-acetyl-L-leucyl-L-leucyl-leucyl-L-norleucinal, decreases the pool of major histocompatibility complex class I-binding peptides and inhibits peptide trimming in the endoplasmic reticulum.
Hughes EA; Ortmann B; Surman M; Cresswell P
Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
J Exp Med, 1996 Apr 1, 183:4, 1569-78
N-acetyl-L-leucyl-L-leucyl-L-norleucinal, (LLnL), which inhibits proteasomes in addition to other proteases, was found to prolong the association of major histocompatibility complex class I molecules with the transporters associated with antigen processing (TAP), and to slow their transport out of the endoplasmic reticulum (ER). LLnL induced a reversible accumulation of ubiquitinated proteins and changed the spectrum of peptides bound by class I molecules. These effects can probably be attributed to proteasome inhibition. Unexpectedly, in the TAP-deficient cell line .174, the rate of intracellular transport of human histocompatibility leukocyte antigen (HLA) A2 was also reduced by LLnL, and the generation of most HLA-A2-associated signal sequence peptides was inhibited. The inhibition of HLA-A2 transport in .174 cells was found to be less sensitive to LLnL than in wild-type cells, and a similar difference was found for a second protease inhibitor, benzyloxycarbonyl-L-leucyl-L-leucyl-L-phenylalanilal. These data suggest that under some conditions such inhibitors can block trimming of peptides by an ER peptidase in addition to inhibiting cytosolic peptide generation.
LA=ENG
96298952
Cysteine Proteinase Inhibitors|*PD; Endoplasmic Reticulum|*ME; Histocompatibility Antigens Class I|*ME; Leupeptins|*PD; Peptides|*ME
Amino Acid Sequence; Biological Transport; Carrier Proteins|ME; Cell Compartmentation; Cytosol|DE; Molecular Sequence Data; Oligopeptides|PD; Protein Binding; Support, Non-U.S. Gov't; Support, U.S. Gov't, P.H.S.
JOURNAL ARTICLE
0022-1007
UNITED STATES
0 (Carrier Proteins); 0 (Cysteine Proteinase Inhibitors); 0 (Histocompatibility Antigens Class I); 0 (Leupeptins); 0 (Oligopeptides); 0 (Peptides); 110044-82-1 (acetylleucyl-leucyl-norleucinal); 143839-79-6 (N-benzyloxycarbonyl-leucyl-leucyl-phenylalaninal)
Record 4 from database: MEDLINE
Resonance energy transfer determination of the distance between the four cysteine-364 residues in Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase.
Alvear M; Encinas MV; Herrera L; Cardemil E
Departamento de Ciencias Qu�imicas, Facultad de Ingenier�ia y Administraci�on, Universidad de la Frontera, Temuco, Chile.
Arch Biochem Biophys, 1994 Mar, 309:2, 231-8
Each of the four subunits of the Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase has one cysteine residue (Cys-364) that is protected against alkylation by MnATP and that is thought to be located at (or close to) the active site (M. Alvear, M. V. Encinas, S. Latshaw, R. G. Kemp, and E. Cardemil, 1992, Biochim. Biophys. Acta 1119, 35-38). To determine the distance relationships between these residues within this tetrameric enzyme, we have derivatized one of these reactive thiols with N-acetyl-N'-(5-sulfo-1-naphthyl) ethylenediamine (AEDANS) and the others progressively with 4-[N-[(acetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole (ANBD). In the doubly labeled protein nonradiative singlet-singlet energy transfer between AEDANS (donor) and ANBD (acceptor) was observed. The efficiency of the energy transfer is proportional to the number of occupied acceptor sites. From these data it has been determined that one of the acceptor sites is 33 A from the donor, and the remaining two sites are 44-46 A from the donor. Cross-linking experiments revealed that mainly cross-linked dimers were produced upon reaction of the enzyme with o-phthalaldehyde and dithiobissuccinimidylpropionate. We interpret these results as an indication that this tetrameric enzyme is most likely composed of an association of two dimers.
LA=ENG
94182934
Cysteine|*CH; Energy Transfer|*; Phosphoenolpyruvate Carboxykinases|*CH; Saccharomyces cerevisiae|*EN
Amino Acid Sequence; Binding Sites; Chemistry, Physical; Cross-Linking Reagents; Fluoresceins; Fluorescent Dyes; Macromolecular Systems; Molecular Sequence Data; Naphthalenesulfonates; Oxadiazoles; Spectrometry, Fluorescence; Spectrophotometry; Sulfhydryl Reagents; Support, Non-U.S. Gov't
JOURNAL ARTICLE
0003-9861
UNITED STATES
EC 4.1.1.32 (Phosphoenolpyruvate Carboxykinases); 0 (Cross-Linking Reagents); 0 (Fluoresceins); 0 (Fluorescent Dyes); 0 (Macromolecular Systems); 0 (Naphthalenesulfonates); 0 (Oxadiazoles); 0 (Sulfhydryl Reagents); 36930-63-9 (1,5-I-AEDANS); 4371-52-2 (Cysteine); 63368-54-7 (5-iodoacetamidofluorescein); 67013-48-3 (4-(N-(iodoacetoxy)ethyl-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole)
SUBSCRIBE: The Wednesday Letter is a free electronic monthly newsletter written and published by Karl Loren. You can view more than 20 back issues of this publication by clicking here. The Wednesday Letter subscription list is maintained on a secure server, no name is ever given or sold to anyone, and it is never used except for this Newsletter. It is automatically published on the Tuesday night just before the first Wednesday of every month. You can subscribe to this free monthly electronic letter by entering your eMail address and name below. You will then automatically receive a request for confirmation, sent to whatever address you have entered. If you do NOT receive this confirmation request, then you will not be subscribed. There may have been an error with your address and you should resubmit. The letter is never sent twice to the same address -- so you do not have to worry about a duplicate subscription. When you receive this confirmation request you must reply to it, or your subscription will not become active. No one can subscribe your name, and address, without you being notified, and if you get an unwanted notice of subscription you only need to DO NOTHING and the subscription will NOT be active.
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You can reach Vibrant Life in many ways, including by mail to Vibrant Life, 1831 N. Bel Aire Drive, Burbank, CA 91504. Within the US and Canada, use the toll free number: (800) 523-4521, the local number: (818) 558-1799, the FAX: (818) 558-7299, eMail to [email protected] or any one of the hundreds of message forms throughout the web site. Vibrant Life normally ships the same day we get an order. There are message forms on each of the 4000+ pages on this site where you can communicate with Vibrant Life. Check out our companion site, at: http://www.oralchelation.net where Karl's 2000 page book is published. Karl Loren is the author and webmaster for this BOOK, as well as for another web site about ORAL CHELATION. He is also the author of a web site about the immune system and COLOSTRUM, as well as FIBROMYALGIA. His personal philosophical articles are at PHILOSOPHY, while his herbal cigarette web site is at INSTEADOF.
Copyright � October 01, 1999 08:27 PM by Vibrant Life, ALL RIGHTS RESERVED. Permission is granted for non-commercial downloading, copying, distribution or redistribution on two conditions: One, that some form of copyright notice is included in every copy distributed or copied, showing the copyright belonging to Vibrant Life, Burbank, CA, at www.oralchelation.com .The second condition is that the material is not to be used for any purpose contrary to the purposes and objectives of this site. This permission does not extend to materials on this site which are copyrighted by others.
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You can reach Vibrant Life in many ways, including by mail to Vibrant Life, 1831 N. Bel Aire Drive, Burbank, CA 91504. Within the US, use the toll free number: (800) 523-4521, the local number: (818) 558-1799, the FAX: (818) 558-7299, eMail to [email protected] or any one of the dozens of message forms throughout the web site. Vibrant Life normally ships the same day we get an order.
Copyright � October 01, 1999 08:27 PM by Vibrant Life, ALL RIGHTS RESERVED. Permission is granted for non-commercial downloading, copying, distribution or redistribution on two conditions: One, that some form of copyright notice is included in every copy distributed or copied, showing the copyright belonging to Vibrant Life, Burbank, CA, at www.oralchelation.com .The second condition is that the material is not to be used for any purpose contrary to the purposes and objectives of this site.