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Butyrylcholinesterase: A Multifaceted Pharmacological Target and Tool

Ha ZY, Mathew S, Yeong KY

Curr Protein Pept Sci, 2020;21(1):99-109.
PMID: 31702488 DOI: 10.2174/1389203720666191107094949

Butyrylcholinesterase is a serine hydrolase that catalyzes the hydrolysis of esters in the body. Unlike its sister enzyme acetylcholinesterase, butyrylcholinesterase has a broad substrate scope and lower acetylcholine catalytic efficiency. The difference in tissue distribution and inhibitor sensitivity also points to its involvement external to cholinergic neurotransmission. Initial studies on butyrylcholinesterase showed that the inhibition of the enzyme led to the increment of brain acetylcholine levels. Further gene knockout studies suggested its involvement in the regulation of amyloid-beta, a brain pathogenic protein. Thus, it is an interesting target for neurological disorders such as Alzheimer's disease. The substrate scope of butyrylcholinesterase was recently found to include cocaine, as well as ghrelin, the "hunger hormone". These findings led to the development of recombinant butyrylcholinesterase mutants and viral gene therapy to combat cocaine addiction, along with in-depth studies on the significance of butyrylcholinesterase in obesity. It is observed that the pharmacological impact of butyrylcholinesterase increased in tandem with each reported finding. Not only is the enzyme now considered an important pharmacological target, it is also becoming an important tool to study the biological pathways in various diseases. Here, we review and summarize the biochemical properties of butyrylcholinesterase and its roles, as a cholinergic neurotransmitter, in various diseases, particularly neurodegenerative disorders.

Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Recombinant Proteins/therapeutic use
Expansion of Hydra actinoporin-like toxin (HALT) gene family: Expression divergence and functional convergence evolved through gene duplication

Yap WY, Tan KJSX, Hwang JS

Toxicon, 2019 Dec;170:10-20.
PMID: 31513812 DOI: 10.1016/j.toxicon.2019.09.007

Hydra actinoporin-like toxin 1 (HALT-1) was previously shown to cause cytolysis and haemolysis in a number of human cells and has similar functional properties to the actinoporins equinatoxin and sticholysin. In addition to HALT-1, five other HALTs (HALTs 2, 3, 4, 6 and 7) were also isolated from Hydra magnipapillata and expressed as recombinant proteins in this study. We demonstrated that recombinant HALTs have cytolytic activity on HeLa cells but each exhibited a different range of toxicity. All six recombinant HALTs bound to sulfatide, while rHALT-1 and rHALT-3 bound to two additional sphingolipids, lysophosphatidic acid and sphingosine-1-phosphate as indicated by the protein-lipid overlay assay. When either tryptophan133 or tyrosine129 of HALT-1 was mutated, the mutant protein lost binding to sulfatide, lysophosphatidic acid and sphingosine-1-phosphate. As further verification of HALTs' binding to sulfatide, we performed ELISA for each HALT. To determine the cell-type specific gene expression of seven HALTs in Hydra, we searched for individual HALT expression in the single-cell RNA-seq data set of Single Cell Portal. The results showed that HALT-1, 4 and 7 were expressed in differentiating stenoteles. HALT-1 and HALT-6 were expressed in the female germline during oogenesis. HALT-2 was strongly expressed in the gland and mucous cells in the endoderm. Information on HALT-3 and HALT-5 could not be found in the single-cell data set. Our findings show that subfunctionalisation of gene expression following duplication enabled HALTs to become specialized in various cell types of the interstitial cell lineage.

Matched MeSH terms: Recombinant Proteins
Fulltext Cytotoxic and apoptosis-inducing effects of wildtype and mutated Hydra actinoporin-like toxin 1 (HALT-1) on various cancer cell lines

Ng TJ, Teo MYM, Liew DS, Effiong PE, Hwang JS, Lim CSY, et al.

PeerJ, 2019;7:e6639.
PMID: 31106043 DOI: 10.7717/peerj.6639

Background: Hydra actinoporin like toxin -1 (HALT-1), is a small 18.5 kDa pore forming toxin derived from Hydra magnipapillata which has been shown to elicit strong haemolytic and cytolytic activity when in contact with cell membranes. Due to its cytotoxic potency, HALT-1 was further investigated for its potential as a toxin moiety candidate in immunotoxin developmental efforts, ideally as a form of targeted therapy against cancer.
Methods: In this study, wtHALT-1 (wild type) and its Y110A mutated binding domain counterpart (mHALT-1) were produced and evaluated for their cytotoxic and apoptotic effects on various cancer cell lines. A total of seven different tumour and non-tumour cell lines including HeLa, HepG2, SW-620, MCF-7, CCD841CoN, NHDF and HCT116 were used. Immunofluorescence assays were used to observe membrane binding and localization changes between both HALT-1 recombinant proteins based on 6xHis-tag detection.
Result: Based on MTT data, mHALT-1 demonstrated a significant reduction of 82% ± 12.21% in cytotoxic activity across all cell lines after the membrane recognition domain had been mutated in comparison to the wtHALT-1. Annexin V FITC/PI assay data also indicated that HeLa, HepG2 and MCF-7 demonstrated an apoptosis-mediated cell death after being treated with wtHALT-1. Additionally, a notable difference between wtHALT-1 and mHALT-1 binding affinity was clearly observed where emission of green fluorescence along the cell membrane was observed only in wtHALT-1 treated cells.
Discussion: These results suggest that mHALT-1 (Y110A) can be potentially developed as a toxin-moiety candidate for the development of future immunotoxins against various human cell-based diseases.

Matched MeSH terms: Recombinant Proteins
Fulltext Dechlorination of polychlorobiphenyl degradation metabolites by a recombinant glutathione S-transferase from Acidovorax sp. KKS102

Shehu D, Alias Z

FEBS Open Bio, 2019 03;9(3):408-419.
PMID: 30868049 DOI: 10.1002/2211-5463.12405

A glutathione S-transferase (GST) with a potential dehalogenation function against various organochlorine substrates was identified from a polychlorobiphenyl (PCB)-degrading organism, Acidovorax sp. KKS102. A homolog of the gene BphK (biphenyl upper pathway K), named BphK-KKS, was cloned, purified and biochemically characterized. Bioinformatic analysis indicated several conserved amino acids that participated in the catalytic activity of the enzyme, and site-directed mutagenesis of these conserved amino acids revealed their importance in the enzyme's catalytic activity. The wild-type and mutant (C10F, K107T and A180P) recombinant proteins displayed wider substrate specificity. The wild-type recombinant GST reacted towards 1-chloro-2,4-dinitrobenzene (CDNB), ethacrynic acid, hydrogen peroxide and cumene hydroperoxide. The mutated recombinant proteins, however, showed significant variation in specific activities towards the substrates. A combination of a molecular docking study and a chloride ion detection assay showed potential interaction with and a dechlorination function against 2-, 3- and 4-chlorobenzoates (metabolites generated during PCB biodegradation) in addition to some organochlorine pesticides (dichlorodiphenyltrichloroethane, endosulfan and permethrin). It was demonstrated that the behavior of the dechlorinating activities varied among the wild-type and mutant recombinant proteins. Kinetic studies (using CDNB and glutathione) showed that the kinetic parameters Km, Vmax, Kcat and Km/Kcat were all affected by the mutations. While C10F and A180P mutants displayed an increase in GST activity and the dechlorination function of the enzyme, the K107T mutant displayed variable results, suggesting a functional role of Lys107 in determining substrate specificity of the enzyme. These results demonstrated that the enzyme should be valuable in the bioremediation of metabolites generated during PCB biodegradation.

Matched MeSH terms: Recombinant Proteins
Evaluation of a Rapid IgG4 Lateral Flow Dipstick Test to Detect Strongyloides stercoralis Infection in Northeast Thailand

Noordin R, Anuar NS, Juri NM, Wongphutorn P, Ruantip S, Kopolrat KY, et al.

Am J Trop Med Hyg, 2021 07 08;105(3):688-691.
PMID: 34237022 DOI: 10.4269/ajtmh.21-0317

Strongyloides stercoralis affects more than half a billion people worldwide, and hyperinfection in immunocompromised patients can be fatal. Elimination of this neglected tropical disease requires field-applicable diagnostic tools. We conducted a laboratory evaluation of a lateral flow rapid dipstick test (SsRapid™) using sera samples from a Strongyloides-endemic area in northeast Thailand. Group 1 was S. stercoralis-positive and larvae- and/or antibody-positive (according to the IgG ELISA) (N = 100). Group 2 had negative fecal examination and IgG ELISA results (N = 25). Group 3 had other parasitic infections and negative IgG ELISA results (N = 25). The results showed good diagnostic sensitivity (82%) and excellent specificity (96%). Suggested improvements in the SsRapid™ test include increased diagnostic sensitivity and conversion to the more robust cassette format. Field studies should be performed as well.

Matched MeSH terms: Recombinant Proteins
Fulltext Effect of O-linked glycosylation on the antigenicity, cellular uptake and trafficking in dendritic cells of recombinant Ber e 1

Jambari NN, Liddell S, Martinez-Pomares L, Alcocer MJC

PLoS One, 2021;16(4):e0249876.
PMID: 33914740 DOI: 10.1371/journal.pone.0249876

Ber e 1, a major Brazil nut allergen, has been successfully produced in the yeast Pichia pastoris expression system as homogenous recombinant Ber e 1 (rBer e 1) with similar physicochemical properties and identical immunoreactivity to its native counterpart, nBer e 1. However, O-linked glycans was detected on the P.pastoris-derived rBer e 1, which is not naturally present in nBer e 1, and may contribute to the allergic sensitisation. In this study, we addressed the glycosylation differences between P. pastoris-derived recombinant Ber e 1 and its native counterparts. We also determined whether this fungal glycosylation could affect the antigenicity and immunogenicity of the rBer e 1 by using dendritic cells (DC) as an immune cell model due to their role in modulating the immune response. We identified that the glycosylation occurs at Ser96, Ser101 and Ser110 on the large chain and Ser19 on the small polypeptide chain of rBer e 1 only. The glycosylation on rBer e 1 was shown to elicit varying degree of antigenicity by binding to different combination of human leukocyte antigens (HLA) at different frequencies compared to nBer e 1 when tested using human DC-T cell assay. However, both forms of Ber e 1 are weak immunogens based from their low response indexes (RI). Glycans present on rBer e 1 were shown to increase the efficiency of the protein recognition and internalization by murine bone marrow-derived dendritic cells (bmDC) via C-type lectin receptors, particularly the mannose receptor (MR), compared to the non-glycosylated nBer e 1 and SFA8, a weak allergenic 2S albumin protein from sunflower seed. Binding of glycosylated rBer e 1 to MR alone was found to not induce the production of IL-10 that modulates bmDC to polarise Th2 cell response by suppressing IL-12 production and DC maturation. Our findings suggest that the O-linked glycosylation by P. pastoris has a small but measurable effect on the in vitro antigenicity of the rBer e 1 compared to its non-glycosylated counterpart, nBer e 1, and thus may influence its applications in diagnostics and immunotherapy.

Matched MeSH terms: Recombinant Proteins/biosynthesis; Recombinant Proteins/immunology; Recombinant Proteins/isolation & purification
Fulltext A Lateral Flow Rapid Test for Human Toxocariasis Developed Using Three Toxocara canis Recombinant Antigens

Yunus MH, Tan Farrizam SN, Abdul Karim IZ, Noordin R

Am J Trop Med Hyg, 2018 Jan;98(1):32-38.
PMID: 29141740 DOI: 10.4269/ajtmh.17-0632

Laboratory diagnosis of toxocariasis is still a challenge especially in developing endemic countries with polyparasitism. In this study, three Toxocara canis recombinant antigens, rTES-26, rTES-30, and rTES-120, were expressed and used to prepare lateral flow immunoglobulin G4 (IgG4) dipsticks. The concordance of the results of the rapid test (comprising three dipsticks) with a commercial IgG-enzyme-linked immunosorbent assay (ELISA) (Cypress Diagnostics, Belgium) was compared against the concordance of two other commercial IgG-ELISA kits (Bordier, Switzerland and NovaTec, Germany) with the Cypress kit. Using Toxocara-positive samples, the concordance of the dipstick dotted with rTES-26, rTES-30, and rTES-120 was 41.4% (12/29), 51.7% (15/29), and 72.4% (21/29), respectively. When positivity with any dipstick was considered as an overall positive rapid test result, the concordance with the Cypress kit was 93% (27/29). Meanwhile, when compared with the results of the Cypress kit, the concordance of IgG-ELISA from NovaTec and Bordier was 100% (29/29) and 89.7% (26/29), respectively. Specific IgG4 has been recognized as a marker of active infection for several helminthic diseases; therefore, the two non-concordant results of the rapid test when compared with the NovaTec IgG-ELISA kit may be from samples of people with non-active infection. All the three dipsticks showed 100% (50/50) concordance with the Cypress kit when tested with serum from individuals who were healthy and with other infections. In conclusion, the lateral flow rapid test is potentially a good, fast, and easy test for toxocariasis. Next, further validation studies and development of a test with the three antigens in one dipstick will be performed.

Matched MeSH terms: Recombinant Proteins
Fulltext Serological diagnostic potential of recombinant outer membrane proteins (rOMPs) from Brucella melitensis in mouse model using indirect enzyme-linked immunosorbent assay

Ahmed IM, Khairani-Bejo S, Hassan L, Bahaman AR, Omar AR

BMC Vet Res, 2015;11:275.
PMID: 26530141 DOI: 10.1186/s12917-015-0587-2

Brucella melitensis is the most important pathogenic species of Brucella spp. which affects goats and sheep and causes caprine and ovine brucellosis, respectively. Serological tests for diagnosis of brucellosis such as Rose Bengal plate test (RBPT) and enzyme-linked immunosorbent assay (ELISA) usually utilize smooth lipopolysaccharides (S-LPS) as a diagnostic antigen which could give false positive serological reactions. Outer membrane proteins (OMP) of B. melitensis have been used as alternative diagnostic antigens rather than S-LPS for differential serological diagnosis of brucellosis, mainly in ELISA with single recombinant OMP (rOMP) as a diagnostic antigen. Nevertheless, the use of single format mainly showed lack of sensitivity against the desired rOMP. Therefore, this study aimed to determine whether a newly developed rOMPs indirect ELISA (rOMPs I-ELISA), based on combination of rOMP25, rOMP28 and rOMP31of B. melitensis, has a potential benefit for use in the serodiagnosis of brucellosis.

Matched MeSH terms: Recombinant Proteins
Fulltext Molecular characterization of a recombinant manganese superoxide dismutase from Lactococcus lactis M4

Tan BH, Chor Leow T, Foo HL, Abdul Rahim R

Biomed Res Int, 2014;2014:469298.
PMID: 24592392 DOI: 10.1155/2014/469298

A superoxide dismutase (SOD) gene of Lactococcus lactis M4 was cloned and expressed in a prokaryotic system. Sequence analysis revealed an open reading frame of 621 bp which codes for 206 amino acid residues. Expression of sodA under T7 promoter exhibited a specific activity of 4967 U/mg when induced with 1 mM of isopropyl-β-D-thiogalactopyranoside. The recombinant SOD was purified to homogeneity by immobilised metal affinity chromatography and Superose 12 gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analyses of the recombinant SOD detected a molecular mass of approximately 27 kDa. However, the SOD was in dimer form as revealed by gel filtration chromatography. The purified recombinant enzyme had a pI of 4.5 and exhibited maximal activity at 25°C and pH 7.2. It was stable up to 45°C. The insensitivity of this lactococcal SOD to cyanide and hydrogen peroxide established that it was a MnSOD. Although it has 98% homology to SOD of L. lactis IL1403, this is the first elucidated structure of lactococcal SOD revealing active sites containing the catalytic manganese coordinated by four ligands (H-27, H-82, D-168, and H-172).

Matched MeSH terms: Recombinant Proteins/metabolism*
Fulltext Engineering the production of major catechins by Escherichia coli carrying metabolite genes of Camellia sinensis

Umar KM, Abdulkarim SM, Radu S, Abdul Hamid A, Saari N

ScientificWorldJournal, 2012;2012:529031.
PMID: 22645428 DOI: 10.1100/2012/529031

A mimicked biosynthetic pathway of catechin metabolite genes from C. sinensis, consisting of flavanone 3 hydroxylase (F3H), dihydroflavonol reductase (DFR), and leucoanthocyanidin reductase (LCR), was designed and arranged in two sets of constructs: (a) single promoter in front of F3H and ribosome-binding sequences both in front of DFR and LCR; (b) three different promoters with each in the front of the three genes and ribosome-binding sequences at appropriate positions. Recombinant E. coli BL (DE3) harbouring the constructs were cultivated for 65 h at 26 °C in M9 medium consisting of 40 g/L glucose, 1 mM IPTG, and 3 mM eriodictyol. Compounds produced were extracted in ethyl acetate in alkaline conditions after 1 h at room temperature and identified by HPLC. Two of the four major catechins, namely, (-)-epicatechin (0.01) and (-)-epicatechin gallate (0.36 mg/L), and two other types ((+)-catechin hydrate (0.13 mg/L) and (-)-catechin gallate (0.04 mg/L)) were successfully produced.

Matched MeSH terms: Recombinant Proteins/chemistry
Brugia malayi infection in Meriones unguiculatus: antibody response to recombinant BmR1

Lim BH, Noordin R, Nor ZM, Rahman RA, Abdullah KA, Sinnadurai S

Exp Parasitol, 2004 Sep-Oct;108(1-2):1-6.
PMID: 15491542

BmR1 recombinant antigen has previously been shown to demonstrate high sensitivity and specificity in the serological diagnosis of brugian filariasis in humans. In this study, the pattern of recognition of antibody to BmR1 during Brugia malayi infection was investigated by employing Meriones unguiculatus as the experimental model. Thirty two gerbils were infected subcutaneously with 120 L(3); and two control groups each comprising 25 animals were employed. ELISA using BmR1 was used to detect filaria-specific IgG antibodies elicited by the gerbils; using sera collected from the day 1 until day 150 post-inoculation (p.i.). The results showed that BmR1 detected B. malayi infection in gerbils harboring adult worms irrespective of the presence of circulating microfilaria, and was exemplified by positive ELISA results in nine a microfilaraemic animals that harbored live adult worms. The initial time of the antibody recognition was at day 8 p.i. and the antibody titre showed some correlation with adult worm burden.

Matched MeSH terms: Recombinant Proteins/immunology
Comparison of the effects between animal-derived trypsin and recombinant trypsin on human skin cells proliferation, gene and protein expression

Manira M, Khairul Anuar K, Seet WT, Ahmad Irfan AW, Ng MH, Chua KH, et al.

Cell Tissue Bank, 2014 Mar;15(1):41-9.
PMID: 23456438 DOI: 10.1007/s10561-013-9368-y

Animal-derivative free reagents are preferred in skin cell culture for clinical applications. The aim of this study was to compare the performance and effects between animal-derived trypsin and recombinant trypsin for skin cells culture and expansion. Full thickness human skin was digested in 0.6 % collagenase for 6 h to liberate the fibroblasts, followed by treatment with either animal-derived trypsin; Trypsin EDTA (TE) or recombinant trypsin; TrypLE Select (TS) to liberate the keratinocytes. Both keratinocytes and fibroblasts were then culture-expanded until passage 2. Trypsinization for both cell types during culture-expansion was performed using either TE or TS. Total cells yield was determined using a haemocytometer. Expression of collagen type I, collagen type III (Col-III), cytokeratin 10, and cytokeratin 14 genes were quantified via RT-PCR and further confirmed with immunocytochemical staining. The results of our study showed that the total cell yield for both keratinocytes and fibroblasts treated with TE or TS were comparable. RT-PCR showed that expression of skin-specific genes except Col-III was higher in the TS treated group compared to that in the TE group. Expression of proteins specific to the two cell types were confirmed by immunocytochemical staining in both TE and TS groups. In conclusion, the performance of the recombinant trypsin is comparable with the well-established animal-derived trypsin for human skin cell culture expansion in terms of cell yield and expression of specific cellular markers.

Matched MeSH terms: Recombinant Proteins/pharmacology*
Biosynthesis and characterization of poly(3-hydroxybutyrate-co-3- hydroxyhexanoate) from palm oil products in a Wautersia eutropha mutant

Loo CY, Lee WH, Tsuge T, Doi Y, Sudesh K

Biotechnol Lett, 2005 Sep;27(18):1405-10.
PMID: 16215858

Palm kernel oil, palm olein, crude palm oil and palm acid oil were used for the synthesis of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] by a mutant strain of Wautersia eutropha (formerly Ralstonia eutropha) harboring the Aeromonas caviae polyhydroxyalkanoate (PHA) synthase gene. Palm kernel oil was an excellent carbon source for the production of cell biomass and P(3HB-co-3HHx). About 87% (w/w) of the cell dry weight as P(3HB-co-3HHx) was obtained using 5 g palm kernel oil/l. Gravimetric and microscopic analyses further confirmed the high PHA content in the recombinant cells. The molar fraction of 3HHx remained constant at 5 mol % regardless of the type and concentration of palm oil products used. The small amount of 3HHx units was confirmed by 13C NMR analysis. The number average molecular weight (M(n)) of the PHA copolymer produced from the various palm oil products ranged from 27 0000 to 46 0000 Da. The polydispersity was in the range of 2.6-3.9.

Matched MeSH terms: Recombinant Proteins/metabolism
Fulltext Analysis of Comparative Sequence and Genomic Data to Verify Phylogenetic Relationship and Explore a New Subfamily of Bacterial Lipases

Masomian M, Rahman RN, Salleh AB, Basri M

PLoS One, 2016;11(3):e0149851.
PMID: 26934700 DOI: 10.1371/journal.pone.0149851

Thermostable and organic solvent-tolerant enzymes have significant potential in a wide range of synthetic reactions in industry due to their inherent stability at high temperatures and their ability to endure harsh organic solvents. In this study, a novel gene encoding a true lipase was isolated by construction of a genomic DNA library of thermophilic Aneurinibacillus thermoaerophilus strain HZ into Escherichia coli plasmid vector. Sequence analysis revealed that HZ lipase had 62% identity to putative lipase from Bacillus pseudomycoides. The closely characterized lipases to the HZ lipase gene are from thermostable Bacillus and Geobacillus lipases belonging to the subfamily I.5 with ≤ 57% identity. The amino acid sequence analysis of HZ lipase determined a conserved pentapeptide containing the active serine, GHSMG and a Ca(2+)-binding motif, GCYGSD in the enzyme. Protein structure modeling showed that HZ lipase consisted of an α/β hydrolase fold and a lid domain. Protein sequence alignment, conserved regions analysis, clustal distance matrix and amino acid composition illustrated differences between HZ lipase and other thermostable lipases. Phylogenetic analysis revealed that this lipase represented a new subfamily of family I of bacterial true lipases, classified as family I.9. The HZ lipase was expressed under promoter Plac using IPTG and was characterized. The recombinant enzyme showed optimal activity at 65 °C and retained ≥ 97% activity after incubation at 50 °C for 1h. The HZ lipase was stable in various polar and non-polar organic solvents.

Matched MeSH terms: Recombinant Proteins/genetics
Cryptocaryon irritans recombinant proteins as potential antigens for sero-surveillance of cryptocaryonosis

Lokanathan Y, Mohd-Adnan A, Kua BC, Nathan S

J Fish Dis, 2016 Sep;39(9):1069-83.
PMID: 27086498 DOI: 10.1111/jfd.12474

Cryptocaryonosis is a major problem for mariculture, and the absence of suitable sero-surveillance tools for the detection of cryptocaryonosis makes it difficult to screen Cryptocaryon irritans-infected fish, particularly asymptomatic fish. In this study, we proposed a serum-based assay using selected C. irritans proteins to screen infected and asymptomatic fish. Eight highly expressed genes were chosen from an earlier study on C. irritans expressed sequence tags and ciliate glutamine codons were converted to universal glutamine codons. The chemically synthesized C. irritans genes were then expressed in an Escherichia coli expression host under optimized conditions. Five C. irritans proteins were successfully expressed in E. coli and purified by affinity chromatography. These proteins were used as antigens in an enzyme-linked immunosorbent assay (ELISA) to screen sera from experimentally immunized fish and naturally infected fish. Sera from both categories of fish reacted equally well with the expressed C. irritans recombinant proteins as well as with sonicated theronts. This study demonstrated the utility of producing ciliate recombinant proteins in a heterologous expression host. An ELISA was successfully developed to diagnose infected and asymptomatic fish using the recombinant proteins as antigens.

Matched MeSH terms: Recombinant Proteins/metabolism
Recombinant long-acting glycoPEGylated factor IX (nonacog beta pegol) in haemophilia B: assessment of target joints in multinational phase 3 clinical trials

Negrier C, Young G, Abdul Karim F, Collins PW, Hanabusa H, Colberg T, et al.

Haemophilia, 2016 Jul;22(4):507-13.
PMID: 26936227 DOI: 10.1111/hae.12902

BACKGROUND: The paradigm(™) 2 and 4 phase 3 clinical trials investigated the safety and efficacy of nonacog beta pegol, a recombinant glycoPEGylated factor IX (FIX) with extended half-life, in previously treated haemophilia B patients.
AIM: These post hoc analyses investigated the bleeding patterns in target joints.
METHODS: Patients randomized to 40 or 10 IU kg(-1) once weekly prophylaxis who had at least one target joint were included. Baseline demographics and disease-specific data were collected. Bleeding patterns were assessed, and an International Society on Thrombosis and Haemostasis (ISTH) definition of target joints was used.
RESULTS: A total of 67% and 8% of patients in the 40 and 10 IU kg(-1) arm, respectively, did not experience target joint bleeds during the paradigm(™) 2 trial. Twenty-four target joints were recorded in each prophylaxis arm at baseline. During the paradigm(™) 2 trial, no bleeds were reported in 17 (71%) and 7 (29%) target joints in the 40 and 10 IU kg(-1) arms respectively. All target joint bleeds in the 40 IU kg(-1) once weekly prophylaxis arm were controlled with a single injection of 40 IU kg(-1) nonacog beta pegol. By the latest ISTH definition, 90% and 58% of target joints in the 40 and 10 IU kg(-1) arms, respectively, were no longer considered target joints at the end of the paradigm(™) 2 trial. At the end of the paradigm(™) 4 extension trial, all target joints in the 40 IU kg(-1) arm were no longer considered target joints.
CONCLUSION: Routine prophylaxis with 40 IU kg(-1) once weekly nonacog beta pegol has the potential for effective management of target joint bleeds in haemophilia B patients.

Matched MeSH terms: Recombinant Proteins/therapeutic use
Production of Toxocara cati TES-120 Recombinant Antigen and Comparison with its T. canis Homolog for Serodiagnosis of Toxocariasis

Zahabiun F, Sadjjadi SM, Yunus MH, Rahumatullah A, Moghaddam MH, Saidin S, et al.

Am J Trop Med Hyg, 2015 Aug;93(2):319-25.
PMID: 26033026 DOI: 10.4269/ajtmh.15-0190

Toxocariasis is a cosmopolitan zoonotic disease caused by the infective larvae of Toxocara canis and T. cati. Diagnosis in humans is usually based on clinical symptoms and serology. Immunoglobulin G (IgG)-enzyme-linked immunosorbent assay kits using T. canis excretory-secretory (TES) larval antigens are commonly used for serodiagnosis. Differences in the antigens of the two Toxocara species may influence the diagnostic sensitivity of the test. In this study, T. cati recombinant TES-120 (rTES-120) was cloned, expressed, and compared with its T. canis homolog in an IgG4-western blot. The diagnostic sensitivity and specificity of T. cati rTES-120 were 70% (33/47) and 100% (39/39), respectively. T. canis rTES-120 showed 57.4% sensitivity and 94.4% specificity. When the results of assays using rTES-120 of both species were considered, the diagnostic sensitivity was 76%. This study shows that using antigens from both Toxocara species may improve the serodiagnosis of toxocariasis.

Matched MeSH terms: Recombinant Proteins/biosynthesis
Isolation and characterization of cDNA clones encoding ADP-glucose pyrophosphorylase from sago palm (Metroxylon sagu)

Au SL, Tan SH, Harikrishna K, Napis S

J. Biochem. Mol. Biol. Biophys., 2002 Oct;6(5):301-8.
PMID: 12385964

Four ADP-glucose pyrophosphorylase cDNA clones were isolated from mature leaves and pith of sago palm by the polymerase chain reaction (PCR) technique. Three of them (agpp10, agpp12 and agpl19) encoded the AGP large subunit, while the fourth clone (agpl1) encoded the small subunit. agpp10 and agpp12 were isolated from pith, agpl19 was isolated from mature leaves, while agpl1 from both tissues. In addition, a full-length cDNA of agpl1 was successfully isolated from a cDNA library of mature leaves by a PCR-based screening technique. Semi-quantitative analysis suggests that agpp10 and agpp12 were detectable only in pith, agpl19 only in leaves, while agpl1 was expressed in both leaves and pith tissues.

Matched MeSH terms: Recombinant Proteins/chemistry
Cloning and sequencing of three new putative toxin genes from Clostridium bifermentans CH18

Barloy F, Lecadet MM, Delécluse A

Gene, 1998 May 12;211(2):293-9.
PMID: 9602158

Three new open reading frames were found downstream from cbm71, a toxin gene from Clostridium bifermentans malaysia (Cbm) strain CH18. The first one (91bp downstream) called cbm72, is 1857bp long and encodes a 71727-Da protein (Cbm72) with a sequence similar to that of Bacillus thuringiensis delta-endotoxins. This protein shows no significant toxicity to mosquito larvae. The two others, cbm17.1 (462bp) and cbm17.2 (459bp), are copies of the same gene encoding Cbm P18 and P16 polypeptides and located 426bp and 1022bp downstream from cbm72, respectively. They encode 17189-Da and 17451-Da proteins with sequences 44.6% similar to that of Aspergillus fumigatus hemolysin; however, they were not hemolytic in the conditions tested.

Matched MeSH terms: Recombinant Proteins/genetics
Serum and salivary IgA antibodies against a defined epitope of the Epstein-Barr virus nuclear antigen (EBNA) are elevated in nasopharyngeal carcinoma

Foong YT, Cheng HM, Sam CK, Dillner J, Hinderer W, Prasad U

Int J Cancer, 1990 Jun 15;45(6):1061-4.
PMID: 1693600

The Epstein-Barr virus nuclear antigen I (EBNA I) is the only latent EBV antigen consistently expressed in malignant tissues of the nasopharynx. A 20-amino-acid synthetic peptide, p107 contains a major epitope of EBNA I. We tested sera from 210 patients with nasopharyngeal carcinoma (NPC) and from 128 normal individuals (NHS) for IgA antibodies to p107 using an enzyme-linked immunosorbent assay (ELISA). Whereas 191/210 (91%) of NPC patients had IgA antibodies to p107, only 17/128 (13.3%) of NHS had such antibodies and only 6/57 (10.5%) of sera from patients with malignancies other than NPC had IgA-p107 reactivity. Thirty-nine salivary samples from 46 NPC patients (84.8%) also contained IgA-p107 antibodies whereas only 3/42 (7.1%) of normal saliva samples were IgA-p107 positive. The results suggest that IgA antibodies to EBNA I may become a useful, easily measurable, marker for NPC.

Matched MeSH terms: Recombinant Proteins/immunology

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