Distinction of cnxH cofactor gene-specified protomers with monoclonal antibodies to Aspergillus nitrate reductase

1988 ◽  
Vol 34 (1) ◽  
pp. 68-72 ◽  
Author(s):  
R. J. Downey ◽  
P. A. South
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Nitrate Reductase ◽  
Aspergillus Nidulans ◽  
Structural Gene ◽  
Molybdenum Cofactor ◽  
Native Enzyme ◽  
Wild Type ◽  
Dimeric Form

The nitrate reductase (NADPH) (EC 1.6.6.3) from Aspergillus nidulans is influenced directly by mutations in the structural gene (niaD) for the major subunit of the enzyme and indirectly by mutation in any of several molybdenum cofactor loci (cnx). The cnxE-14 and the cnxH-3 mutants have been noted to contain the enzyme in two distinct forms following induction with nitrate. With the cnxH-3 as a prototype cnxH mutant, 10 other cnxH were found to be devoid of the assembled (dimeric) form of the enzyme. Two monoclonal antibodies specific for the native enzyme of the wild type (biA-1) recognized an epitope on the enzyme from the cnxE-14 and cnxH-3 mutants that was common to both and another that was unique to the cnxH gene specified protomer. Another monoclonal antibody recognized an epitope that occurs only in the assembled dimeric form of the enzyme from the wild type or the cnxE-14 mutant. The experiments further substantiate the cnxH phenotype as one involving unassembled protomers of the nitrate reductase in Aspergillus.

Regulation of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AldDH) in Aspergillus nidulans

1983 ◽  
Vol 217 (1208) ◽  
pp. 243-264 ◽  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Aspergillus Nidulans ◽  
Structural Gene ◽  
Aldehyde Dehydrogenase ◽  
Gel Electrophoresis ◽  
Regulatory Gene ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Wild Type ◽  
Cell Extracts

There is a single major alcohol dehydrogenase (ADH) and a single major aldehyde dehydrogenase (AldDH) in Aspergillus nidulans . Both ADH and AldDH are induced by ethanol and by acetaldehyde and both are subject to carbon catabolite repression. ADH and AldDH are necessary for the utilization of ethanol and of threonine, indicating that both compounds are utilized via acetaldehyde. ADH and AldDH each give a single major activity band on gel electrophoresis. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of cell extracts shows at least two similar ADH polypeptides of approximate relative molecular mass (r. m. m.) 41000 and two similar AldDH polypeptides of approximate r. m. m. 57000. The in vitro translation of mRNA from induced, carbon derepressed wild-type cells gives up to three ADH polypeptides in the r. m. m. range 39000-43000 and an AldDH polypeptide of approximate r. m. m. 57000. The mRNA from uninduced, carbon repressed wild-type cells does not direct the synthesis of the ADH and AldDH polypeptides. This indicates that the regulation of ADH and AldDH is at the level of transcription and/or post-transcriptional modification. The probable explanation of the multiple ADH polypeptides is post-transcriptional modification of the mRNA. Allyl alcohol mutants were made by using diepoxyoctane and γ-rays as mutagens. There are two classes, alcA and alcR . Neither class can utilize ethanol or threonine as a carbon source. The alcA mutants lack normal ADH and are recessive. Of the 47 alcA mutants examined 39 do not make the ADH polypeptides while eight do so. Therefore alcA is the structural gene for ADH. The two alcA mutants tested do not make functional mRNA for ADH. The alcR mutants lack both ADH and AldDH and are recessive. No alcR mutants make the ADH or the AldDH polypeptides. The three alcR mutants tested do not make functional ADH or AldDH mRNA. The mutant alcR 125 is a nonsense mutant, which establishes that alcR codes for a protein. The alcA and alcR genes are adjacent on chromosome VII and a preliminary fine-structure map of the alcA gene has been made. Three mutants that cannot utilize ethanol or threonine and have ADH, but lack AldDH, define a gene AldA on chromosome VIII. The aldA 23 mutant makes the AldDH polypeptides, the other two aldA mutants do not. Therefore aldA is probably the structural gene for AldDH. Our current hypothesis is that alcA and aldA are the structural genes for ADH and AldDH respectively and alcR is a transacting regulatory gene coding for a protein whose function is necessary for the expression of the alcA and aldA genes.

scholarly journals Genetics of nitrate reductase inUstilago maydis

1970 ◽  
Vol 16 (2) ◽  
pp. 151-163 ◽  
Author(s):  
C. M. Lewis ◽  
J. R. S. Fincham
Keyword(s):  
Nitrate Reductase ◽  
Structural Gene ◽  
Nitrate Reduction ◽  
Ustilago Maydis ◽  
Xanthine Dehydrogenase ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Sensitive Mutant ◽  
Temperature Sensitive Mutant ◽  
Complementation Groups

SUMMARYMutants ofUstilago maydishave been isolated both, deficient and derepressed for nitrate reduction. Those deficient in enzyme fall into six groups, one of which is the structural gene. Enzyme which has proved to be more labile than that of wild-type has been isolated from a temperature-sensitive mutant at this locus. All the mutants in the structural gene have xanthine dehydrogenase activity and the situation closely parallels that ofAspergillus nidulans.The derepressed mutants fall into four complementation groups and all are partially derepressed in that they are further inducible by nitrate. Full derepression can be conferred by induction of a second mutation. In one analysed case the second reinforcing mutation proved to be pheno-typically similar to the first one when separated from it.

scholarly journals Epitope Switching as a Novel Escape Mechanism of HIV to CCR5 Monoclonal Antibodies

2010 ◽  
Vol 54 (2) ◽  
pp. 734-741 ◽  
Author(s):  
Andreas Jekle ◽  
Milloni Chhabra ◽  
Adriane Lochner ◽  
Sonja Meier ◽  
Eugene Chow ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Resistant Virus ◽  
Extracellular Loop ◽  
Wild Type ◽  
Hiv Resistance ◽  
Ccr5 Receptor ◽  
N Terminus ◽  
Loop 2 ◽  
Virus Strains

ABSTRACT In passaging experiments, we isolated HIV strains resistant to MAb3952, a chemokine (C-C motif) receptor 5 (CCR5) monoclonal antibody (MAb) that binds to the second extracellular domain (extracellular loop 2 [ECL-2]) of CCR5. MAb3952-resistant viruses remain CCR5-tropic and are cross-resistant to a second ECL-2-specific antibody. Surprisingly, MAb3952-resistant viruses were more susceptible to RoAb13, a CCR5 antibody binding to the N terminus of CCR5. Using CCR5 receptor mutants, we show that MAb3952-resistant virus strains preferentially use the N terminus of CCR5, while the wild-type viruses preferentially use ECL-2. We propose this switch in the CCR5 binding site as a novel mechanism of HIV resistance.

Differential effects of a molybdopterin synthase sulfurylase (moeB) mutation on Escherichia coli molybdoenzyme maturation

2002 ◽  
Vol 80 (4) ◽  
pp. 435-443 ◽  
Author(s):  
Damaraju Sambasivarao ◽  
Raymond J Turner ◽  
Peter T Bilous ◽  
Richard A Rothery ◽  
Gillian Shaw ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Mutant Strain ◽  
Physiological Function ◽  
Signal Sequence ◽  
Molybdenum Cofactor ◽  
Wild Type ◽  
Dmso Reductase ◽  
E Coli ◽  
Membrane Bound

We have generated a chromosomal mutant of moeB (moeBA228T) that demonstrates limited molybdenum cofactor (molybdo-bis(molybdopterin guanine dinucleotide) (Mo-bisMGD)) availability in Escherichia coli and have characterized its effect on the maturation and physiological function of two well-characterized respiratory molybdoenzymes: the membrane-bound dimethylsulfoxide (DMSO) reductase (DmsABC) and the membrane-bound nitrate reductase A (NarGHI). In the moeBA228T mutant strain, E. coli F36, anaerobic respiratory growth is possible on nitrate but not on DMSO, indicating that cofactor insertion occurs into NarGHI but not into DmsABC. Fluorescence analyses of cofactor availability indicate little detectable cofactor in the moeBA228T mutant compared with the wild-type, suggesting that NarGHI is able to scavenge limiting cofactor, whereas DmsABC is not. MoeB functions to sulfurylate MoaD, and in the structure of the MoeB–MoaD complex, Ala-228 is located in the interface region between the two proteins. This suggests that the moeBA228T mutation disrupts the interaction between MoeB and MoaD. In the case of DmsABC, despite the absence of cofactor, the twin-arginine signal sequence of DmsA is cleaved in the moeBA228T mutant, indicating that maturation of the holoenzyme is not cofactor-insertion dependent.Key words: mdybdenum cofactor, DMSO reductase, nitrate reductase.

scholarly journals Sensitivity of SARS-CoV-2 Variants to Neutralization by Convalescent Sera and a VH3-30 Monoclonal Antibody

2021 ◽  
Vol 12 ◽  
Author(s):  
Shuai Yue ◽  
Zhirong Li ◽  
Yao Lin ◽  
Yang Yang ◽  
Mengqi Yuan ◽  
...  
Keyword(s):  
South Africa ◽  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Binding Activity ◽  
Wild Type ◽  
The Past ◽  
Global Pandemic ◽  
Neutralizing Capacity ◽  
Novel Coronavirus ◽  
Medical Countermeasures

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of novel coronavirus disease (COVID-19). Though vaccines and neutralizing monoclonal antibodies (mAbs) have been developed to fight COVID-19 in the past year, one major concern is the emergence of SARS-CoV-2 variants of concern (VOCs). Indeed, SARS-CoV-2 VOCs such as B.1.1.7 (UK), B.1.351 (South Africa), P.1 (Brazil), and B.1.617.1 (India) now dominate the pandemic. Herein, we found that binding activity and neutralizing capacity of sera collected from convalescent patients in early 2020 for SARS-CoV-2 VOCs, but not non-VOC variants, were severely blunted. Furthermore, we observed evasion of SARS-CoV-2 VOCs from a VH3-30 mAb 32D4, which was proved to exhibit highly potential neutralization against wild-type (WT) SARS-CoV-2. Thus, these results indicated that SARS-CoV-2 VOCs might be able to spread in convalescent patients and even harbor resistance to medical countermeasures. New interventions against these SARS-CoV-2 VOCs are urgently needed.

Association of the NAD(P)H-bispecific nitrate reductase structural gene with the Nar7 locus in barley

Genome ◽  
1995 ◽  
Vol 38 (4) ◽  
pp. 743-746 ◽  
Author(s):  
R. L. Warner ◽  
D. A. Kudrna ◽  
A. Kleinhofs
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Nitrate Reductase ◽  
Structural Gene ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Reductase Activity ◽  
Wild Type ◽  
Restriction Fragment Length

The NADH-specific and NAD(P)H-bispecific nitrate reductase genes from barley have been cloned and sequenced. To determine if the Nar7 locus encodes the NAD(P)H-bispecific nitrate reductase structural gene, a cross was made between a wild-type cultivar, Morex (Nar7 Nar7), and Az70 (nar7w nar7w), a mutant from the cultivar Steptoe that is deficient in NAD(P)H-bispecific nitrate reductase activity. A probe specific to the NAD(P)H-bispecific nitrate reductase structural gene detected restriction fragment length polymorphism between the parents. This probe was used to classify selected F2 progeny for restriction fragment length genotype. All the NAD(P)H nitrate reductase deficient F2 progeny (24/101) possessed the Az70 restriction fragment genotype. The absence of recombination between the NAD(P)H-bispecific nitrate reductase deficient genotype and the NAD(P)H-bispecific nitrate reductase restriction fragment length genotype indicates that the two traits are closely associated in inheritance and that Nar7 is probably the NAD(P)H-bispecific nitrate reductase structural gene.Key words: Hordeum vulgare, nitrate reductase, restriction fragment length polymorphism.

The efficacy of antiepidermal growth factor receptor monoclonal antibodies for patients with KRAS G13D mutations and chemorefractory metastatic colorectal cancer.

2012 ◽  
Vol 30 (4_suppl) ◽  
pp. 527-527
Author(s):  
Kohei Akiyoshi ◽  
Yasuhide Yamada ◽  
Naoki Takahashi ◽  
Yoshitaka Honma ◽  
Satoru Iwasa ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Growth Factor ◽  
Metastatic Colorectal Cancer ◽  
Growth Factor Receptor ◽  
Cancer Center ◽  
Wild Type ◽  
Duration Of Treatment ◽  
Kras Mutations

527 Background: The treatment benefits of epidermal growth factor receptor (EGFR) monoclonal antibodies for patients with KRAS mutations have not been demonstrated. However, some studies have suggested that all KRAS mutations are not equivalent, and that KRAS G13D mutations might have some survival benefit. Methods: We retrospectively analyzed the efficacy and toxicity of treatment with EGFR monoclonal antibody in 8 patients with KRAS G13D mutations and 5-FU/oxaliplatin/irinotecan (CPT) refractory metastatic colorectal cancer compared with 94 KRAS wild type patients at the National Cancer Center Hospital. Results: Eight patients with KRAS G13D mutations were treated with anti-EGFR monoclonal antibodies between July, 2009 and July, 2011. The median age was 66 (42-70); male/female 6/2; PS was 0/1/2, 2/5/1; treatment regimen was cetuximab/ cetuximab+CPT/ panitumumab+CPT, 2/5/1. Response rate (RR) was 12.5% and disease control rate (DCR) was 50.0% with 1 PR, 3 SD, and 4 PD. The PR case treated with cetuximab+CPT showed marked regression of tumor and long duration of treatment (9 months). The progression free survival (PFS) of 2 SD cases was 4.2 and 3.9 months. The other SD case is now on treatment. The median PFS of the 8 patients was 2.1 months (95% confidence interval [CI]: 0.0-5.2). The median overall survival (OS) has not been reached. Grade 3/4 toxicities included 1 hypomagnesemia G4 and 1 rash acneiform G3. Meanwhile, 94 KRAS wild type patients treated with anti EGFR monoclonal antibodies had an RR of 22.3% and DCR was 66.0% with 21 PR, 41 SD, 30 PD, and 2 NE. PFS was 5.6 months (95% CI: 4.9-6.3) and OS was 8.6 months (95% CI: 6.5-10.7). Conclusions: In this analysis, we identified one PR to anti-EGFR monoclonal antibody in a patient with KRAS G13D mutation and chemo-refractory metastatic colorectal cancer. However, we were unable to demonstrate equivalent efficacy in patients with KRAS G13D mutations and KRAS wild type patients. Further studies are needed to evaluate the efficacy and prognosis for this treatment.

scholarly journals Resistance to anti-EGFR targeted therapy mediated by oncogenetic mutations in colorectal cancer: Revision of the dogma?

2020 ◽  
pp. 2-2
Keyword(s):  
Monoclonal Antibody ◽  
Epidermal Growth Factor Receptor ◽  
Monoclonal Antibodies ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Colorectal Carcinoma ◽  
Tumor Cells ◽  
Growth Factor Receptor ◽  
Wild Type ◽  
Epidermal Growth

Traditionally, systemic treatment for high stage colorectal carcinoma (CRC) is mainly fluorouracil-based chemotherapy [1]. The anti-epidermal growth factor receptor (EGFR) monoclonal antibodies, by acting on specific molecular pathways in tumor growth or modulating immune response towards tumor cells, provide a more targeted response, a better side effect profile and greater impact on patient survival compared with conventional molecules. This monoclonal antibody that binds the extracellular domain of epidermal growth factor receptor, is known to be effective only in a subset of KRAS wild-type colorectal cancers. Patients with mutations in either KRAS or NRAS gene are not eligible for anti-EGFR monoclonal antibody therapy [3]. This is due to downstream activation of the Ras/Raf/MAPK pathway by mutated RAS protein, leading to cell proliferation which cannot be sufficiently inhibited by anti-EGFR receptor monoclonal antibodies [4]. With the increasing choices of targeted agents, more and more biomarkers are tested. Currently, the standard recommended biomarker panel for colorectal carcinoma would include KRAS, NRAS, BRAF gene hotspot mutation detection and microsatellite instability test [5]. With the advances in genomic profiling and sequencing and the understanding of the resistance mechanisms, the contraindication of anti-EGFR therapy in mutant KRAS patients may be revised. Based on the fact that the KRAS mutation in CRC suppresses the phosphorylation of the AMP-activated protein kinase (AMPK) known to be toxic for the tumor cells, Hua et al obtained a satisfactory response to for the anti-EGFR antibodies in mutant KRAS CRC xenograft models after reactivation of the AMPK [6]. Knickelbein et al demonstrated that the anti-EGFR antibodies induce the death of CRC cells via a p73- dependent transcriptional activation of the pro-apoptotic Bcl-2 family protein (PUMA). This action is abolished in case of KRAS mutation. These authors admitted that the restoration of this pathway by inhibiting aurora kinases preferentially kills mutant KRAS CRC cells and overcomes KRAS-mediated resistance to anti-EGFR antibodies [7]. In fifty-one CRC patient-derived xenografts study, Lee et al showed that KRAS mutants expressed remarkably elevated autocrine levels of high-affinity EGFR ligands compared with wild-type KRAS. The use of an anti-EGFR IgG1 antibody that displays potent inhibitory effects on highaffinity EGFR ligand enhanced CRC KRAS mutant cells cytotoxicity [8]. Dealing with the resistance to targeted therapies in CRC patient looks feasible. It could allow to broaden the indications of anti-EGFR therapy and provide a better survival for a larger group of CRC patients. In this era of precision and personalized medicine, a complete case specific tumor profiling and the comprehensive study of the tumorogenesis mechanisms should allow to overcome the intrinsic and acquired resistance to these targeted high effective therapies.

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