scholarly journals Thermostable and Long-Circulating Albumin-Conjugated Arthrobacter globiformis Urate Oxidase

Pharmaceutics ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1298 ◽  
Author(s):  
Byungseop Yang ◽  
Inchan Kwon
Keyword(s):  
Enzymatic Activity ◽  
Tumor Lysis Syndrome ◽  
Alder Reaction ◽  
Urate Oxidase ◽  
Circulation Time ◽  
Arthrobacter Globiformis ◽  
Wild Type ◽  
Computational Stability ◽  
Site Specific

Urate oxidase derived from Aspergillus flavus has been investigated as a treatment for tumor lysis syndrome, hyperuricemia, and gout. However, its long-term use is limited owing to potential immunogenicity, low thermostability, and short circulation time in vivo. Recently, urate oxidase isolated from Arthrobacter globiformis (AgUox) has been reported to be thermostable and less immunogenic than the Aspergillus-derived urate oxidase. Conjugation of human serum albumin (HSA) to therapeutic proteins has become a promising strategy to prolong circulation time in vivo. To develop a thermostable and long-circulating urate oxidase, we investigated the site-specific conjugation of HSA to AgUox based on site-specific incorporation of a clickable non-natural amino acid (frTet) and an inverse electron demand Diels–Alder reaction. We selected 14 sites for frTet incorporation using the ROSETTA design, a computational stability prediction program, among which AgUox containing frTet at position 196 (Ag12) exhibited enzymatic activity and thermostability comparable to those of wild-type AgUox. Furthermore, Ag12 exhibited a high HSA conjugation yield without compromising the enzymatic activity, generating well-defined HSA-conjugated AgUox (Ag12-HSA). In mice, the serum half-life of Ag12-HSA was approximately 29 h, which was roughly 17-fold longer than that of wild-type AgUox. Altogether, this novel formulated AgUox may hold enhanced therapeutic efficacy for several diseases.

scholarly journals Developmental aspects of adipose tissue in GH receptor and prolactin receptor gene disrupted mice: site-specific effects upon proliferation, differentiation and hormone sensitivity

2006 ◽  
Vol 191 (1) ◽  
pp. 101-111 ◽  
Author(s):  
David J Flint ◽  
Nadine Binart ◽  
Stephanie Boumard ◽  
John J Kopchick ◽  
Paul Kelly
Keyword(s):  
Adipose Tissue ◽  
Receptor Gene ◽  
Metabolic Effects ◽  
Wild Type ◽  
Extrinsic Factors ◽  
Differentiation Potential ◽  
Site Specific ◽  
Proliferation And Differentiation

Direct metabolic effects of GH on adipose tissue are well established, but effects of prolactin (PRL) have been more controversial. Recent studies have demonstrated PRL receptors on adipocytes and effects of PRL on adipose tissue in vitro. The role of GH in adipocyte proliferation and differentiation is also controversial, since GH stimulates adipocyte differentiation in cell lines, whereas it stimulates proliferation but inhibits differentiation of adipocytes in primary cell culture. Using female gene disrupted (ko) mice, we showed that absence of PRL receptors (PRLRko) impaired development of both internal and s.c. adipose tissue, due to reduced numbers of adipocytes, an effect differing from that of reduced food intake, where cell volume is decreased. In contrast, GHRko mice exhibited major decreases in the number of internal adipocytes, whereas s.c. adipocyte numbers were increased, even though body weight was decreased by 40–50%. The changes in adipose tissue in PRLRko mice appeared to be entirely due to extrinsic factors since preadipocytes proliferated and differentiated in similar fashion to wild-type animals in vitro and their response to insulin and isoproterenol was similar to wild-type animals. This contrasted with GHRko mice, where s.c. adipocytes proliferated, differentiated, and responded to hormones in identical fashion to controls, whereas parametrial adipocytes exhibited markedly depressed proliferation and differentiation potential and failed to respond to insulin or noradrenaline. Our results provide in vivo evidence that both GH and PRL stimulate differentiation of adipocytes but that the effects of GH are site specific and induce intrinsic changes in the precursor population, which are retained in vitro.

Recombinant Variants of Tissue-type Plasminogen Activator Containing Amino Acid Substitutions in the Fibronectin Finger-like Domain and the Kringle 1 Domain

1994 ◽  
Vol 72 (06) ◽  
pp. 893-899
Author(s):  
Hitoshi Yahara ◽  
Keiji Matsumoto ◽  
Hiroyuki Maruyama ◽  
Tetsuya Nagaoka ◽  
Yasuhiro Ikenaka ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Half Life ◽  
Bolus Injection ◽  
Tissue Type ◽  
Clot Lysis ◽  
Wild Type ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Plasma Half Life

SummaryTissue-type plasminogen activator (t-PA) is a fibrin-specific agent which is used to treat acute myocardial infarction. Pharmacokinetic-ally, t-PA is characterized by a rapid clearance from the circulation. In a previous study, we constructed variant forms of t-PA with genetic modifications at the fibronectin finger-like domain (finger domain) or at the kringle 1 domain (K1 domain). The finger modified variant, t-PA N37S.S38V.G39V.R40E. A41F.Q42S had about a 6.0-fold higher plasma half-life in vivo than wild-type t-PA. Two variants with modifications in the K1 domain, t-PA G161R.K162R.S165W and t-PA N115P, showed an improved kinetic parameters and a 2.2-fold higher plasma half-life in vivo than wild-type t-PA, respectively. To create a recombinant variant of t-PA with a higher enzymatic activity and a further prolonged half-life in vivo, the genes containing each modifications were joined and expressed in animal cells. The two variants, t-PA N37S.S38V G39V.R40E.A41F.Q42S.G161R.K162R.S165W and t-PA N37S.S38V.G39V.R40E.A41F.Q42S.N 115P, were purified from conditioned media and their biochemical, pharmacokinetic and thrombolytic profiles were investigated. Although the variant t-PA N37S.S38V.G39V.R40E.A41F.Q42S.G161R.K162R.S165W demonstrated an impaired enzymatic activity compared to the wild:type t-PA, the half-life of the variant, t-PA N37S.S38V.G39V.R40E.A41F.Q42S. N115P, following intravenous bolus injection in rabbits was considerably longer than that of finger-domain modified variants. Human plasma clot lysis assay estimated the fibrinolytic activity of both variants to be about 2.0-fold less effective than that of the wild-type t-PA. In the rabbit jugular vein clot lysis model, doses of 1.0 and 0.0625 mg/kg were required for about 70% lysis in the wild-type t-PA and t-PA N37S.S38V.G39V.R40E.A41F.Q42S.N115P, respectively. These findings suggested that the variant in this study can be used at a lower dosage in a single bolus injection.

scholarly journals Chemical Modification of Cysteine with 3-Arylpropriolonitrile Improves the In Vivo Stability of Albumin-Conjugated Urate Oxidase Therapeutic Protein

Biomedicines ◽  
2021 ◽  
Vol 9 (10) ◽  
pp. 1334
Author(s):  
Byungseop Yang ◽  
Inchan Kwon
Keyword(s):  
Half Life ◽  
Late Phase ◽  
Hydrolytic Stability ◽  
Therapeutic Protein ◽  
Urate Oxidase ◽  
Cysteine Modification ◽  
Reaction Products ◽  
Site Specific

3-arylpropiolonitriles (APN) are promising alternatives to maleimide for chemo-selective thiol conjugation, because the reaction product has a remarkably hydrolytic stability compared with that of thiol-maleimide reactions in vitro. However, whether cysteine modification with APN enhances stability in vivo compared to thiol-maleimide reactions remains unclear, probably due to the too short in vivo serum half-life of a protein to observe significant cleavage of thiol-maleimide/-APN reaction products. The conjugation of human serum albumin (HSA) to a therapeutic protein reportedly prolongs the in vivo serum half-life. To evaluate the in vivo stability of the thiol-APN reaction product, we prepared HSA-conjugated Arthrobacter globiformis urate oxidase (AgUox), a therapeutic protein for gout treatment. Site-specific HSA conjugation to AgUox was achieved by combining site-specific incorporation of tetrazine containing an amino acid (frTet) into AgUox and a crosslinker containing trans-cyclooctene and either thiol-maleimide (AgUox-MAL-HSA) or -APN chemistry (AgUox-APN-HSA). Substantial cleavage of the thioester of AgUox-MAL-HSA was observed in vitro, whereas no cleavage of the thiol-APN product of AgUox-APN-HSA was observed. Furthermore, the in vivo serum half-life of AgUox-APN-HSA in the late phase was significantly longer than that of AgUox-MAL-HSA. Overall, these results demonstrate that the thiol-APN chemistry enhanced the in vivo stability of the HSA-conjugated therapeutic protein.

scholarly journals Mucosal Adjuvanticity and Immunogenicity of LTR72, a Novel Mutant of Escherichia coli Heat-labile Enterotoxin with Partial Knockout of ADP-ribosyltransferase Activity

1998 ◽  
Vol 187 (7) ◽  
pp. 1123-1132 ◽  
Author(s):  
Marzia Monica Giuliani ◽  
Giuseppe Del Giudice ◽  
Valentina Giannelli ◽  
Gordon Dougan ◽  
Gill Douce ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Enzymatic Activity ◽  
Wild Type ◽  
Ileal Loop ◽  
Heat Labile ◽  
A Subunit ◽  
Reduced Toxicity ◽  
Adp Ribosyltransferase

Heat-labile Escherichia coli enterotoxin (LT) has the innate property of being a strong mucosal immunogen and adjuvant. In the attempt to reduce toxicity and maintain the useful immunological properties, several LT mutants have been produced. Some of these are promising mucosal adjuvants. However, so far, only those that were still toxic maintained full adjuvanticity. In this paper we describe a novel LT mutant with greatly reduced toxicity that maintains most of the adjuvanticity. The new mutant (LTR72), that contains a substitution Ala → Arg in position 72 of the A subunit, showed only 0.6% of the LT enzymatic activity, was 100,000-fold less toxic than wild-type LT in Y1 cells in vitro, and was at least 20 times less effective than wild-type LT in the rabbit ileal loop assay in vivo. At a dose of 1 μg, LTR72 exhibited a mucosal adjuvanticity, similar to that observed with wild-type LT, better than that induced by the nontoxic, enzymatically inactive LTK63 mutant, and much greater than that of the recombinant B subunit. This trend was consistent for both the amounts and kinetics of the antibody induced, and priming of antigen-specific T lymphocytes. The data suggest that the innate high adjuvanticity of LT derives from the independent contribution of the nontoxic AB complex and the enzymatic activity. LTR72 optimizes the use of both properties: the enzymatic activity for which traces are enough, and the nontoxic AB complex, the effect of which is dose dependent. In fact, in dose–response experiments in mice, 20 μg of LTR72 were a stronger mucosal adjuvant than wild-type LT. This suggests that LTR72 may be an excellent candidate to be tested in clinical trials.

scholarly journals Lambda Xis Degradation In Vivo by Lon and FtsH

1998 ◽  
Vol 180 (6) ◽  
pp. 1573-1577 ◽  
Author(s):  
Gerald G. Leffers ◽  
Susan Gottesman
Keyword(s):  
Half Life ◽  
Double Mutant ◽  
Bacterial Chromosome ◽  
Multicopy Plasmid ◽  
Nonpermissive Temperature ◽  
Phage Lambda ◽  
Wild Type ◽  
Site Specific ◽  
In The Wild

ABSTRACT Lambda Xis, which is required for site-specific excision of phage lambda from the bacterial chromosome, has a much shorter functional half-life than Int, which is required for both integration and excision (R. A. Weisberg and M. E. Gottesman, p. 489–500,in A. D. Hershey, ed., The Bacteriophage Lambda, 1971). We found that Xis is degraded in vivo by two ATP-dependent proteases, Lon and FtsH (HflB). Xis was stabilized two- to threefold more than in the wild type in a lon mutant and as much as sixfold more in a lon ftsH double mutant at the nonpermissive temperature for the ftsH mutation. Integration of lambda into the bacterial chromosome was delayed in thelon ftsH background, suggesting that accumulation of Xis in vivo interferes with integration. Overexpression of Xis in wild-type cells from a multicopy plasmid inhibited integration of lambda and promoted curing of established lysogens, confirming that accumulation of Xis interferes with the ability of Int to establish and maintain an integrated prophage.

scholarly journals Site-Specific 111In-Radiolabeling of Dual-PEGylated Porous Silicon Nanoparticles and Their In Vivo Evaluation in Murine 4T1 Breast Cancer Model

Pharmaceutics ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 686 ◽  
Author(s):  
Dave Lumen ◽  
Simo Näkki ◽  
Surachet Imlimthan ◽  
Elisavet Lambidis ◽  
Mirkka Sarparanta ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Porous Silicon ◽  
Silicon Nanoparticles ◽  
Click Reaction ◽  
Circulation Time ◽  
In Vivo Evaluation ◽  
Site Specific ◽  
Porous Silicon Nanoparticles ◽  
4T1 Breast Cancer

Polyethylene glycol (PEG) has been successfully used for improving circulation time of several nanomaterials but prolonging the circulation of porous silicon nanoparticles (PSi NPs) has remained challenging. Here, we report a site specific radiolabeling of dual-PEGylated thermally oxidized porous silicon (DPEG-TOPSi) NPs and investigation of influence of the PEGylation on blood circulation time of TOPSi NPs. Trans-cyclooctene conjugated DPEG-TOPSi NPs were radiolabeled through a click reaction with [111In]In-DOTA-PEG4-tetrazine (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and the particle behavior was evaluated in vivo in Balb/c mice bearing 4T1 murine breast cancer allografts. The dual-PEGylation significantly prolonged circulation of [111In]In-DPEG-TOPSi particles when compared to non-PEGylated control particles, yielding 10.8 ± 1.7% of the injected activity/g in blood at 15 min for [111In]In-DPEG-TOPSi NPs. The improved circulation time will be beneficial for the accumulation of targeted DPEG-TOPSi to tumors.

scholarly journals Site-Specific Recombination of Bacteriophage P22 Does Not Require Integration Host Factor

1999 ◽  
Vol 181 (14) ◽  
pp. 4245-4249 ◽  
Author(s):  
Eun Hee Cho ◽  
Chan-Eun Nam ◽  
Renato Alcaraz ◽  
Jeffrey F. Gardner
Keyword(s):  
Integration Host Factor ◽  
Host Factor ◽  
Maximum Efficiency ◽  
Wild Type ◽  
Bacteriophage P22 ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Mutant Cells ◽  
Formation Of Complexes

ABSTRACT Site-specific recombination by phages λ and P22 is carried out by multiprotein-DNA complexes. Integration host factor (IHF) facilitates λ site-specific recombination by inducing DNA bends necessary to form an active recombinogenic complex. Mutants lacking IHF are over 1,000-fold less proficient in supporting λ site-specific recombination than wild-type cells. Although the attPregion of P22 contains strong IHF binding sites, in vivo measurements of integration and excision frequencies showed that infecting P22 phages can perform site-specific recombination to its maximum efficiency in the absence of IHF. In addition, a plasmid integration assay showed that integrative recombination occurs equally well in wild-type and ihfA mutant cells. P22 integrative recombination is also efficient in Escherichia coli in the absence of functional IHF. These results suggest that nucleoprotein structures proficient for recombination can form in the absence of IHF or that another factor(s) can substitute for IHF in the formation of complexes.

scholarly journals A comparative analysis among ADAR mutant mice reveals site-specific regulation of RNA editing

2019 ◽  
Author(s):  
Pedro Henrique Costa Cruz ◽  
Yuki Kato ◽  
Taisuke Nakahama ◽  
Toshiharu Shibuya ◽  
Yukio Kawahara
Keyword(s):  
Rna Editing ◽  
Mutant Mouse ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Simple Method ◽  
Site Specific ◽  
Specific Manner ◽  
Specific Regulation ◽  
A Site

ABSTRACTAdenosine-to-inosine RNA editing is an essential posttranscriptional modification catalyzed by adenosine deaminase acting on RNA (ADAR)1 and ADAR2 in mammals. For numerous sites in coding sequences (CDS) and microRNAs (miRNAs), editing is highly conserved and has significant biological consequences, for example, by altering amino acid residues and target recognition. However, technical limitations have prevented a comprehensive and quantitative study to determine how specific ADARs contribute to each site. Here, we developed a simple method in which each RNA region with an editing site was amplified separately and combined for deep sequencing. Using this method, we compared the editing ratios of all sites that were either definitely or possibly conserved in CDS and miRNAs in the cerebral cortex and spleen of wild-type mice, Adar1E861A/E861AIfih−/− mice expressing inactive ADAR1 (Adar1 KI) and Adar2−/−Gria2R/R (Adar2 KO) mice. We found that the editing ratio was frequently upregulated in either Adar mutant mouse strain. In contrast, we found that the presence of both ADAR1 and ADAR2 was required for the efficient editing of specific sites. In addition, some sites, such as miR-3099-3p, showed no preference for either ADAR. We further created double mutant Adar1 KI Adar2 KO mice and observed viable and fertile animals with complete absence of editing, suggesting that ADAR1 and ADAR2 are the sole enzymes responsible for all editing sites in vivo. Collectively, these findings indicate that editing is regulated in a site-specific manner by the different interplay between ADAR1 and ADAR2.

scholarly journals Point Mutations in the Integron Integrase IntI1 That Affect Recombination and/or Substrate Recognition

1998 ◽  
Vol 180 (20) ◽  
pp. 5437-5442 ◽  
Author(s):  
Annie Gravel ◽  
Nancy Messier ◽  
Paul H. Roy
Keyword(s):  
Dna Binding ◽  
Antibiotic Resistance Gene ◽  
Point Mutations ◽  
Wild Type ◽  
Conserved Residues ◽  
Site Specific ◽  
Gene Cassettes ◽  
In Vivo Recombination

ABSTRACT The site-specific recombinase IntI1 found in class 1 integrons catalyzes the excision and integration of mobile gene cassettes, especially antibiotic resistance gene cassettes, with a site-specific recombination system. The integron integrase belongs to the tyrosine recombinase (phage integrase) family. The members of this family, exemplified by the lambda integrase, do not share extensive amino acid identities, but three invariant residues are found within two regions, designated box I and box II. Two conserved residues are arginines, one located in box I and one in box II, while the other conserved residue is a tyrosine located at the C terminus of box II. We have analyzed the properties of IntI1 variants carrying point mutations at the three conserved residues of the family in in vivo recombination and in vitro substrate binding. We have made four proteins with mutations of the conserved box I arginine (R146) and three mutants with changes of the box II arginine (R280); of these, MBP-IntI1(R146K) and MBP-IntI1(R280K) bind to the attI1 site in vitro, but only MBP-IntI1(R280K) is able to excise cassettes in vivo. However, the efficiency of recombination and DNA binding for MBP-IntI1(R280K) is lower than that obtained with the wild-type MBP-IntI1. We have also made two proteins with mutations of the tyrosine residue (Y312), and both mutant proteins are similar to the wild-type fusion protein in their DNA-binding capacity but are unable to catalyze in vivo recombination.

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