scholarly journals Cloning, sequencing and expression of the pyrophosphate-dependent phosphofructo-1-kinase from Naegleria fowleri

1995 ◽  
Vol 307 (1) ◽  
pp. 143-149 ◽  
Author(s):  
K L Wessberg ◽  
S Skolnick ◽  
J Xu ◽  
F Marciano-Cabral ◽  
R G Kemp
Keyword(s):  
Nucleotide Binding ◽  
Binding Motif ◽  
Naegleria Fowleri ◽  
Reading Frame ◽  
Site Characteristic ◽  
High Conservation ◽  
Phosphofructokinase Activity ◽  
Cloned Cdna ◽  
Sequencing And Expression ◽  
Nægleria Fowleri

The cDNA for the PPi-dependent phosphofructo-1-kinase has been cloned and sequenced from a cDNA library prepared from the free-living amoeba Naegleria fowleri. The coding sequence of the cDNA consists of 1311 bases which translates into 437 amino acids with a molecular mass of 48095 Da. Comparison of the sequence with those of the previously described sequences of PPi-dependent phosphofructokinases from Propionibacterium freudenreichii and potato tuber revealed amino acid identities of 23 and 28% respectively and high conservation in those regions assumed to be part of the active site. The reading frame was cloned into an expression vector, which was transformed into Escherichia coli. Extracts of the transformed cells contained PPi-dependent phosphofructokinase activity that could be purified to homogeneity. The activity was lost on incubation with the chaotropic agent, KSCN, and recovered by subsequent incubation with AMP. These properties are consistent with those described by Mertens, De Jonckheere and Van Schaftingen [Biochem. J. (1993) 292, 797-803] for the enzyme prepared from Naegleria and support the idea that the cloned cDNA coded for the complete native enzyme. No nucleotide-binding motif or evidence for a nucleotide-binding site characteristic of the ATP-dependent phosphofructokinases could be found within the primary structure.

Cloning, sequencing, and expression of Na(+)-H+ antiporter cDNAs from human tissues

1992 ◽  
Vol 262 (4) ◽  
pp. C1069-C1076 ◽  
Author(s):  
K. Takaichi ◽  
D. Wang ◽  
D. F. Balkovetz ◽  
D. G. Warnock
Keyword(s):  
Plasma Membranes ◽  
Mutant Mouse ◽  
Human Kidney ◽  
Kidney Cortex ◽  
Human Tissues ◽  
Specific Expression ◽  
Reading Frame ◽  
Mouse Fibroblasts ◽  
Cloned Cdna ◽  
Sequencing And Expression

Two types of Na(+)-H+ antiporter with different sensitivities to amiloride analogues have been identified in mammalian plasma membranes. A human Na(+)-H+ antiporter cDNA was obtained by Sardet and co-workers (C. Sardet, L. Counillon, A. Franchi, and J. Pouyssegur. Cell 56: 271-280, 1989) using mutant mouse fibroblasts lacking Na(+)-H+ antiporter transformed with human genomic DNA. However, the amiloride sensitivity of this cloned Na(+)-H+ antiporter was not precisely determined. Furthermore, the reported cDNA sequence may be a chimera of human and mouse genes. Hence we isolated a Na(+)-H+ antiporter cDNA actually expressed in human tissues and characterized its amiloride sensitivity. Our 4 kb cDNA obtained from human kidney cortex contained the identical open reading frame to that previously reported and the entire 3' terminus, which was quite different from that reported. This discrepancy was not due to differences in tissue-specific expression because cDNAs from different human tissues were identical, and single bands were observed under high stringency on Northern blots of various human tissues. Na(+)-H+ antiporter activity of mutant mouse fibroblasts deficient in Na(+)-H+ antiporter activity transfected with the cloned cDNA was very sensitive to amiloride and 5-N substituted analogues of amiloride. Thus the cloned cDNA represents the NHE-1 isoform of the Na(+)-H+ antiporter.

scholarly journals Dipeptidyl peptidase III is a zinc metallo-exopeptidase: Molecular cloning and expression

1998 ◽  
Vol 329 (2) ◽  
pp. 275-282 ◽  
Author(s):  
Katsuhiko FUKASAWA ◽  
M. Kayoko FUKASAWA ◽  
Makoto KANAI ◽  
Shingo FUJII ◽  
Junzo HIROSE ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dipeptidyl Peptidase ◽  
Cloning And Expression ◽  
Zinc Binding ◽  
Binding Motif ◽  
Ph Optimum ◽  
Reading Frame ◽  
Theoretical Molecular Mass ◽  
Cloned Cdna ◽  
Zinc Binding Domain

We have purified dipeptidyl peptidase III (EC 3.4.14.4) from human placenta. It had a pH optimum of 8.8 and readily hydrolysed Arg-Arg-β-naphthylamide. Monoamino acid-, Gly-Phe-, Gly-Pro- and Bz-Arg-β-naphthylamides were not hydrolysed at all. The enzyme was inhibited by p-chloromercuriphenylsulphonic acid, metal chelators and 3,4-dichloroisocoumarin and contained 1 mol of zinc per mol of enzyme. The zinc dissociation constant was 250 fM at pH 7.4 as determined by the zinc binding study. We isolated, by immunological screening of a Uni-ZAP XR cDNA library constructed from rat liver mRNA species, a cDNA clone with 2633 bp encoding the rat enzyme. The longest open reading frame encodes a 827-residue protein with a theoretical molecular mass of 92790 Da. Escherichia coli SOLR cells were infected with the pBluescript phagemid containing the cloned cDNA and established the overexpression of a protein that hydrolysed Arg-Arg-β-naphthylamide. The recombinant protein was purified and the amino acid sequence of the protein was confirmed. We presumed that the putative zinc-binding domain involved in catalysis was present in the recombinant enzyme. It was a novel zinc-binding motif in that one amino acid residue was inserted into the conserved HEXXH motif characteristic of the metalloproteinases.

scholarly journals Expression of the nfa1 Gene Cloned from Pathogenic Naegleria fowleri in Nonpathogenic N. gruberi Enhances Cytotoxicity against CHO Target Cells In Vitro

2005 ◽  
Vol 73 (7) ◽  
pp. 4098-4105 ◽  
Author(s):  
Seok-Ryoul Jeong ◽  
Sang-Chul Lee ◽  
Kyoung-Ju Song ◽  
Sun Park ◽  
Kyongmin Kim ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Target Cells ◽  
Naegleria Fowleri ◽  
Specific Primers ◽  
Reading Frame ◽  
Reverse Transcription Pcr ◽  
Nægleria Fowleri ◽  
Green Fluorescent ◽  
Naegleria Gruberi

ABSTRACT The pathogenic amoeba Naegleria fowleri has a 360-bp nfa1 gene that encodes the Nfa1 protein (13.1 kDa), which is located in the pseudopodia of the amoeba, and an anti-Nfa1 antibody reduces N. fowleri-induced mammalian-cell cytotoxicity in vitro. In contrast, an anti-Nfa1 antibody cannot detect Nfa1 protein expression in the nonpathogenic amoeba Naegleria gruberi, which also possesses the nfa1 gene. In the present study, the nfa1 gene cloned from pathogenic N. fowleri was transfected into nonpathogenic N. gruberi to determine whether it was related to pathogenicity. The nfa1 gene was initially inserted into a eukaryotic transfection vector, pEGFP-C2, containing a cytomegalovirus promoter and the green fluorescent protein (GFP) gene, and was designed as pEGFP-C2/nfa1UTR (nfa1UTR contains 5′ upstream regions, the nfa1 open reading frame, and 3′ downstream regions). After transfection, the green fluorescence was observed in the cytoplasm of N. gruberi trophozoites. These transfectants were preserved for more than 9 months after selection. The transfected nfa1 gene was observed by PCR using nfa1- and vector-specific primers in the genomic DNA of N. gruberi transfected with the pEGFP-C2/nfa1UTR vector. In addition, the nfa1 and GFP genes were identified by reverse transcription-PCR in transgenic N. gruberi. The Nfa1 protein expressed in transgenic N. gruberi was identified as a 13.1-kDa band by Western blotting using an anti-Nfa1 antibody. Finally, N. gruberi transfected with the pEGFP-C2/nfa1UTR vector was found to have enhanced cytotoxicity against CHO cells compared with naïve N. gruberi.

Efficacy of a combined system of copper and silver and free chlorine for inactivation of Naegleria fowleri amoebas in water

1995 ◽  
Vol 31 (5-6) ◽  
pp. 119-122 ◽  
Author(s):  
J. M. Cassells ◽  
M. T. Yahya ◽  
C. P. Gerba ◽  
J. B. Rose
Keyword(s):  
Synergistic Effect ◽  
Exposure Time ◽  
Silver Ions ◽  
Free Chlorine ◽  
Log10 Reduction ◽  
Naegleria Fowleri ◽  
Combined System ◽  
Water Ph ◽  
Nægleria Fowleri ◽  
Time Required

Electrolytically generated copper and silver ions (400:40 and 800:80 μg/l) were evaluated, separately and combined with 1.0 mg/l free chlorine, for their efficacy in reducing the viable numbers of Naegleria fowleri amoebas in water (pH 7.3 and 23-25°C). Inactivation rates (k = log10 reduction/min) and T99 values (exposure time required to achieve a 99% or a 2 log10 reduction) of the disinfectants were determined. Copper and silver alone, at ratio of 400:40 to 800:80 μg/l caused no significant inactivation of N. fowleri even after 72 hours of exposure (k = 0.00017 and 0.00013, respectively). Addition of 1.0 mg/l free chlorine to water which contained 400:40 or 800:80 μg/l copper and silver resulted in enhanced inactivation rates (k = 0.458 and 0.515, respectively) compared to either chlorine alone (k = 0.33) or the metals alone. Water containing 800:80 μg/l copper and silver with 1.0 mg/l chlorine showed a T99 value of 3.9 minutes, while chlorine alone showed a T99 of 6.1 minutes. Enhanced inactivation of N. fowleri by a combined system of free chlorine and copper and silver may be attributed to the different mechanism that each disinfectant utilizes in inactivating the amoebas, and may suggest a synergistic effect.

scholarly journals A Novel Cysteine Protease Inhibitor of Naegleria fowleri That Is Specifically Expressed during Encystation and at Mature Cysts

Pathogens ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 388
Author(s):  
Hương Giang Lê ◽  
A-Jeong Ham ◽  
Jung-Mi Kang ◽  
Tuấn Cường Võ ◽  
Haung Naw ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Cysteine Protease ◽  
Expression Profiles ◽  
Critical Role ◽  
Cyst Formation ◽  
Cysteine Proteases ◽  
Cysteine Protease Inhibitor ◽  
Naegleria Fowleri ◽  
Nægleria Fowleri ◽  
Cystatin Family

Naegleria fowleri is a free-living amoeba that is ubiquitous in diverse natural environments. It causes a fatal brain infection in humans known as primary amoebic meningoencephalitis. Despite the medical importance of the parasitic disease, there is a great lack of knowledge about the biology and pathogenicity of N. fowleri. In this study, we identified and characterized a novel cysteine protease inhibitor of N. fowleri (NfCPI). NfCPI is a typical cysteine protease inhibitor belonging to the cystatin family with a Gln-Val-Val-Ala-Gly (QVVAG) motif, a characteristic motif conserved in the cystatin family of proteins. Bacterially expressed recombinant NfCPI has a dimeric structure and exhibits inhibitory activity against several cysteine proteases including cathespin Bs of N. fowleri at a broad range of pH values. Expression profiles of nfcpi revealed that the gene was highly expressed during encystation and cyst of the amoeba. Western blot and immunofluorescence assays also support its high level of expression in cysts. These findings collectively suggest that NfCPI may play a critical role in encystation or cyst formation of N. fowleri by regulating cysteine proteases that may mediate encystation or mature cyst formation of the amoeba. More comprehensive studies to investigate the roles of NfCPI in encystation and its target proteases are necessary to elucidate the regulatory mechanism and the biological significance of NfCPI.

Transmission of Naegleria fowleri between Mice

1983 ◽  
Vol 69 (1) ◽  
pp. 249 ◽  
Author(s):  
R. G. May ◽  
D. T. John
Keyword(s):  
Naegleria Fowleri ◽  
Nægleria Fowleri

Development of a rapid DNA extraction method and one-step nested PCR for the detection of Naegleria fowleri from the environment

2011 ◽  
Vol 45 (16) ◽  
pp. 5211-5217 ◽  
Author(s):  
Arine Fadzlun Ahmad ◽  
James Lonnen ◽  
Peter W. Andrew ◽  
Simon Kilvington
Keyword(s):  
Dna Extraction ◽  
Nested Pcr ◽  
Extraction Method ◽  
Naegleria Fowleri ◽  
Dna Extraction Method ◽  
One Step ◽  
Nægleria Fowleri
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