Biochemical and Pharmacokinetic Properties of PEGylated Cystathionine γ-Lyase from Aspergillus carneus KF723837

2015 ◽  
Vol 25 (5) ◽  
pp. 301-310 ◽  
Author(s):  
Ashraf S.A. El-Sayed ◽  
Marwa A. Yassin ◽  
Salwa A. Khalaf ◽  
Mohamed El-Batrik ◽  
Gul Shad Ali ◽  
...  
Keyword(s):  
Specific Activity ◽  
Covalent Binding ◽  
Catalytic Efficiency ◽  
Life Time ◽  
Negative Control ◽  
Native Page ◽  
Pharmacokinetic Properties

Cystathionine &#947;-lyase (CGL) was purified to its electrophoretic homogeneity from<i> Aspergillus carneus</i> by various chromatographic approaches. The purified enzyme has four identical subunits of 52 kDa based on SDS and native PAGE analyses. To improve its structural stability, purified CGL was modified by covalent binding to polyethylene glycol moieties. The specific activity of free-CGL and PEG-CGL was 59.71 and 48.71 U/mg, respectively, with a PEGylation yield of 81.5 and 70.7% modification of surface ε-amino groups. Free- and modified CGL have the same pattern of pH stability (8.0-9.0). At 50°C, the thermal stability [half-life time (T<sub>1/2</sub>)] of PEG-CGL was increased by 40% in comparison to free-CGL. The activity of CGL was completely inhibited by hydroxylamine and Hg<sup>+2</sup>, with no effect by EDTA. Free-CGL (0.04 m<smlcap>M</smlcap><sup>-1</sup>s<sup>-1</sup>) and PEG-CGL (0.03 m<smlcap>M</smlcap><sup>-1</sup>s<sup>-1</sup>) have a similar catalytic efficiency to <smlcap>L</smlcap>-cystathionine as a substrate. The inhibition constant values of propargylglycine were 0.31 and 0.52 µ<smlcap>M</smlcap> for the free- and PEG-CGL, respectively. By in vitro proteolysis, PEG-CGL retains >50% of its initial activity compared to <10% of the free-CGL for acid protease for 30 min. From in vivo pharmacokinetics in New Zealand white rabbits, the T<sub>1/2</sub> was 19.1 and 28.9 h for the Holo free-CGL and PEG-CGL, respectively, ensuring the role of PEGylation on shielding the CGL surface from proteolytic attack, reducing its antigenicity, and stabilizing its internal Schiff base. By external infusion of pyridoxal 5′-phosphate (10 µ<smlcap>M</smlcap>), the T<sub>1/2</sub> of free- and PEG-CGL was prolonged to 24 and 33 h, respectively, so dissociation of pyridoxal 5′-phosphate was one of the main causes of loss of enzyme activity. The biochemical and hematological responses of rabbits to free- and PEG-CGL were assessed, with relative similarity to the negative control, confirming the nil toxicity of enzymes. The titer of IgG was duplicated in response to free- versus PEG-CGL after 45 days. To the best of our knowledge, this is the first report concerned with purification and PEGylation of CGL from fungi, with higher affinity for <smlcap>L</smlcap>-cystathionine. With further molecular studies, CGL will be a promising enzyme against various cardiovascular diseases and antioxidant deficiency, as well as for generation of a neurotransmitter (H<sub>2</sub>S).

scholarly journals Impact of GPR1 signaling on maternal high-fat feeding and placenta metabolism in mice

2019 ◽  
Vol 316 (6) ◽  
pp. E987-E997 ◽  
Author(s):  
Binbin Huang ◽  
Chen Huang ◽  
Huashan Zhao ◽  
Wen Zhu ◽  
Baobei Wang ◽  
...  
Keyword(s):  
Biological Effects ◽  
Negative Control ◽  
Feedback Mechanism ◽  
High Fat ◽  
Fatty Acid Binding ◽  
Maternal Metabolism ◽  
Phosphorylated Akt

Chemerin and G protein-coupled receptor 1 (GPR1) are increased in serum and placenta in mice during pregnancy. Interestingly, we observed increased serum chemerin levels and decreased GPR1 expression in placenta of high-fat-diet-fed mice compared with chow-fed mice at gestational day 18. GPR1 protein and gene levels were significantly decreased in gestational diabetes mellitus (GDM) patient placentas. Therefore, we hypothesized that chemerin/GPR1 signaling might participate in the pathogenic mechanism of GDM. We investigated the role of GPR1 in carbohydrate homeostasis during pregnancy using pregnant mice transfected with small interfering RNA for GPR1 or a negative control. GPR1 knockdown exacerbated glucose intolerance, disrupted lipid metabolism, and decreased β-cell proliferation and insulin levels. Glucose transport protein-3 and fatty acid binding protein-4 were downregulated with reducing GPR1 in vivo and in vitro via phosphorylated AKT pathway. Taken together, our findings first demonstrate the expression of GPR1, the characterization of its direct biological effects in humans and mice, as well as the molecular mechanism that indicates the role of GPR1 signaling in maternal metabolism during pregnancy, suggesting a novel feedback mechanism to regulate glucose balance during pregnancy, and GPR1 could be a potential target for the detection and therapy of GDM.

scholarly journals Anticancer Ruthenium(III) Complexes and Ru(III)-Containing Nanoformulations: An Update on the Mechanism of Action and Biological Activity

2019 ◽  
Vol 12 (4) ◽  
pp. 146 ◽  
Author(s):  
Claudia Riccardi ◽  
Domenica Musumeci ◽  
Marco Trifuoggi ◽  
Carlo Irace ◽  
Luigi Paduano ◽  
...  
Keyword(s):  
Mechanism Of Action ◽  
Anticancer Agents ◽  
Biomedical Applications ◽  
Special Focus ◽  
Anticancer Properties ◽  
Pharmacokinetic Properties ◽  
Pharmacological Potential

The great advances in the studies on metal complexes for the treatment of different cancer forms, starting from the pioneering works on platinum derivatives, have fostered an increasingly growing interest in their properties and biomedical applications. Among the various metal-containing drugs investigated thus far, ruthenium(III) complexes have emerged for their selective cytotoxic activity in vitro and promising anticancer properties in vivo, also leading to a few candidates in advanced clinical trials. Aiming at addressing the solubility, stability and cellular uptake issues of low molecular weight Ru(III)-based compounds, some research groups have proposed the development of suitable drug delivery systems (e.g., taking advantage of nanoparticles, liposomes, etc.) able to enhance their activity compared to the naked drugs. This review highlights the unique role of Ru(III) complexes in the current panorama of anticancer agents, with particular emphasis on Ru-containing nanoformulations based on the incorporation of the Ru(III) complexes into suitable nanocarriers in order to enhance their bioavailability and pharmacokinetic properties. Preclinical evaluation of these nanoaggregates is discussed with a special focus on the investigation of their mechanism of action at a molecular level, highlighting their pharmacological potential in tumour disease models and value for biomedical applications.

Negative control of basophil expansion by IRF-2 critical for the regulation of Th1/Th2 balance

Blood ◽  
2005 ◽  
Vol 106 (6) ◽  
pp. 2011-2017 ◽  
Author(s):  
Shigeaki Hida ◽  
Masumi Tadachi ◽  
Takashi Saito ◽  
Shinsuke Taki
Keyword(s):  
Negative Control ◽  
Interleukin 4 ◽  
Th2 Polarization ◽  
Kit Gene ◽  
Th2 Development ◽  
Neutral Conditions ◽  
Th2 Differentiation

Abstract Although basophils are known to produce interleukin 4 (IL-4), the roles of these cells have been documented only in mice infected with parasites or in the effector phase of allergic inflammations. Here we show that naive mice lacking the transcription factor, interferon regulatory factor 2 (IRF-2), exhibited signal transducer and activator of transcription 6 (Stat6)–independent expansion of basophils in the periphery. IRF-2 appeared to act autonomously in the cells to negatively regulate the expansion of, but not cytokine production by, basophils. Spontaneous Th2 polarization of CD4+ T cells was observed in these mice and the genetic reduction of basophil numbers by mutating the Kit gene abolished such a polarization in vivo. We also found that both basophils and IL-4 derived from them were indeed essential for Th2 development under neutral conditions in vitro. Furthermore, neutralization of IL-3 abolished IL-4 production by basophils during Th1/Th2 differentiation cultures and subsequent Th2 development. These results indicated that basophils acted as a cellular converter to turn the neutral IL-3 into the Th2-inducing IL-4 during the initiation of Th1/Th2 differentiation. Thus, the negative regulatory role of IRF-2 on the basophil population size is critically important for preventing excess Th2 polarization and the Th1/Th2 balance in naive animals.

scholarly journals Rational design of a highly efficient catalytic system for the production of 3′-phosphoadenosine-5′-phosphosulfate from ATP

2021 ◽  
Author(s):  
Kaifang Liu ◽  
Xiulai Chen ◽  
Yunlu Zhong ◽  
Jia Liu ◽  
Guipeng Hu ◽  
...  
Keyword(s):  
Catalytic System ◽  
Rational Design ◽  
Specific Activity ◽  
Catalytic Efficiency ◽  
Disodium Salt ◽  
In Vivo Testing ◽  
Dynamics Simulations ◽  
Rate Limiting

The compound 3′-phosphoadenosine-5′-phosphosulfate (PAPS) serves as a sulfate group donor in the production of valuable sulfated compounds, such as glycosaminoglycan and oxamniquine. However, elevated costs and low conversion efficiency limit the industrial applicability of PAPS. Here, we designed and constructed an efficient and controllable catalytic system for the conversion of ATP (disodium salt) into PAPS without inhibition from by-products. In vitro and in vivo testing in Escherichia coli identified adenosine-5′-phosphosulfate kinase from Penicillium chrysogenum (PcAPSK) as the rate-limiting enzyme. Based on analysis of the catalytic steps and molecular dynamics simulations, a mechanism-guided “ADP expulsion” strategy was developed to generate an improved PcAPSK variant (L7), with a specific activity of 48.94 U·mg-1 and 73.27-fold higher catalytic efficiency (kcat/Km) that of the wild-type enzyme. The improvement was attained chiefly by reducing the ADP-binding affinity of PcAPSK, as well as by changing the enzyme’s flexibility and lid structure to a more open conformation. By introducing PcAPSK L7 in an in vivo catalytic system, 73.59 mM (37.32 g·L-1) PAPS was produced from 150 mM ATP in 18.5 h using a 3-L bioreactor. The achieved titer is the highest reported to date and corresponds to a 98.13% conversion rate. The proposed strategy will facilitate industrial production of PAPS as well as the engineering of similar enzymes.

scholarly journals 14-3-3 Facilitates Ras-Dependent Raf-1 Activation In Vitro and In Vivo

1998 ◽  
Vol 18 (7) ◽  
pp. 3947-3955 ◽  
Author(s):  
Sandrine Roy ◽  
Robert A. McPherson ◽  
Ann Apolloni ◽  
Jun Yan ◽  
Annette Lane ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Specific Activity ◽  
Membrane Recruitment ◽  
Oncogenic Ras ◽  
Epidermal Growth

ABSTRACT 14-3-3 proteins complex with many signaling molecules, including the Raf-1 kinase. However, the role of 14-3-3 in regulating Raf-1 activity is unclear. We show here that 14-3-3 is bound to Raf-1 in the cytosol but is totally displaced when Raf-1 is recruited to the plasma membrane by oncogenic mutant Ras, in vitro and in vivo. 14-3-3 is also displaced when Raf-1 is targeted to the plasma membrane. When serum-starved cells are stimulated with epidermal growth factor, some recruitment of 14-3-3 to the plasma membrane is evident, but 14-3-3 recruitment correlates with Raf-1 dissociation and inactivation, not with Raf-1 recruitment. In vivo, overexpression of 14-3-3 potentiates the specific activity of membrane-recruited Raf-1 without stably associating with the plasma membrane. In vitro, Raf-1 must be complexed with 14-3-3 for efficient recruitment and activation by oncogenic Ras. Recombinant 14-3-3 facilitates Raf-1 activation by membranes containing oncogenic Ras but reduces the amount of Raf-1 that associates with the membranes. These data demonstrate that the interaction of 14-3-3 with Raf-1 is permissive for recruitment and activation by Ras, that 14-3-3 is displaced upon membrane recruitment, and that 14-3-3 may recycle Raf-1 to the cytosol. A model that rationalizes many of the apparently discrepant observations on the role of 14-3-3 in Raf-1 activation is proposed.

scholarly journals The Biological Potential Hidden in Inclusion Bodies

Pharmaceutics ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 157 ◽  
Author(s):  
Laia Gifre-Renom ◽  
Joaquin Seras-Franzoso ◽  
Diana Rafael ◽  
Fernanda Andrade ◽  
Olivia Cano-Garrido ◽  
...  
Keyword(s):  
Protein Delivery ◽  
Inclusion Bodies ◽  
Room Temperature ◽  
Specific Activity ◽  
Covalent Binding ◽  
Biological Potential ◽  
Model Protein ◽  
Metalloproteinase 9

Inclusion bodies (IBs) are protein nanoclusters obtained during recombinant protein production processes, and several studies have demonstrated their potential as biomaterials for therapeutic protein delivery. Nevertheless, IBs have been, so far, exclusively sifted by their biological activity in vitro to be considered in further protein-based treatments in vivo. Matrix metalloproteinase-9 (MMP-9) protein, which has an important role facilitating the migration of immune cells, was used as model protein. The MMP-9 IBs were compared with their soluble counterpart and with MMP-9 encapsulated in polymeric-based micelles (PM) through ionic and covalent binding. The soluble MMP-9 and the MMP-9-ionic PM showed the highest activity values in vitro. IBs showed the lowest activity values in vitro, but the specific activity evolution in 50% bovine serum at room temperature proved that they were the most stable format. The data obtained with the use of an air-pouch mouse model showed that MMP-9 IBs presented the highest in vivo activity compared to the soluble MMP-9, which was associated only to a low and a transitory peak of activity. These results demonstrated that the in vivo performance is the addition of many parameters that did not always correlate with the in vitro behavior of the protein of interest, becoming especially relevant at evaluating the potential of IBs as a protein-based nanomaterial for therapeutic purposes.

scholarly journals The role of somatostatin and dopamine D2 receptors in endocrine tumors

2011 ◽  
Vol 18 (6) ◽  
pp. R233-R251 ◽  
Author(s):  
Federico Gatto ◽  
Leo J Hofland
Keyword(s):  
Pituitary Adenomas ◽  
Hormone Secretion ◽  
Somatostatin Receptors ◽  
Somatostatin Analogs ◽  
Negative Control ◽  
Endocrine Tumors ◽  
Wide Group

Somatostatin (SS) and dopamine (DA) receptors have been highlighted as two critical regulators in the negative control of hormonal secretion in a wide group of human endocrine tumors. Both families of receptors belong to the superfamily of G protein-coupled receptors and share a number of structural and functional characteristics. Because of the generally reported high expression of somatostatin receptors (SSTRs) in neuroendocrine tumors (NET), somatostatin analogs (SSA) have a pronounced role in the medical therapy for this class of tumors, especially pituitary adenomas and well-differentiated gastroenteropancreatic NET (GEP NET). Moreover, NET express not only SSTR but also frequently dopamine receptors (DRs), and DA agonists targeting the D2receptor (D2) have been demonstrated to be effective in controlling hormone secretion and cell proliferation inin vivoandin vitrostudies. The treatment with SSAs combined with DA agonists has already been demonstrated efficacious in a subgroup of patients with GH-secreting pituitary adenomas and few reported cases of carcinoids. The recent availability of new selective and universal SSA and DA agonists, as well as the chimeric SS/DA compounds, may shed new light on the potential role of SSTR and D2as combined targets for biotherapy in NET. This review provides an overview of the latest studies evaluating the expression of SSTR and DR in NET, focusing on their co-expression and the possible clinical implications of such co-expression. Moreover, the most recent insights in SSTR and D2pathophysiology and the future perspectives for treatment with SSA, DA agonists, and SS/DA chimeric compounds are discussed.

Role of 25-hydroxyvitamin D3 dose in determining rat 1,25-dihydroxyvitamin D3 production

1990 ◽  
Vol 258 (5) ◽  
pp. E780-E789 ◽  
Author(s):  
R. Vieth ◽  
K. McCarten ◽  
K. H. Norwich
Keyword(s):  
Specific Activity ◽  
Metabolic Clearance ◽  
Dihydroxyvitamin D3 ◽  
Hydroxyvitamin D ◽  
Dihydroxyvitamin D ◽  
25 Hydroxyvitamin D ◽  
Dose Dependent

To understand the relationships among 1) the dose of 25-hydroxyvitamin D [25(OH)D] in vivo, 2) the activity of 1-hydroxylase in renal mitochondria, and 3) the production of 1,25-dihydroxyvitamin D [1,25(OH)2D] in vivo, we gave rats different chronic or acute doses of 25-hydroxyvitamin D3 [25(OH)D3]. We followed the metabolism of intracardially administered [25-hydroxy-26,27-methyl-3H]cholecalciferol [25(OH)[3H]D3] for 24 h before killing by measuring extracts of serum by chromatography. Specific activity of 1-hydroxylase in kidney was measured at death. In rats given 0-2,000 pmol 25(OH)D3 chronically by mouth, there was a dose-dependent decline in the percent of serum radioactivity made up of 1,25-dihydroxy-[26,27-methyl-3H]cholecalciferol [1,25(OH)2[3H]D3] as well as a decline in mitochondrial 1-hydroxylase, and these correlated significantly (r = 0.83, P less than 0.001). Serum %1,25(OH)2[3H]D3 in this experiment ranged from 0.8 to 42%. A small part of this range could be accounted for by a faster metabolic clearance rate (MCR) of 1,25(OH)2D3 from rats supplemented with 25(OH)D3 (MCR, 2.12 +/- 0.10 ml/min) compared with rats restricted in vitamin D (MCR, 0.94 +/- 0.06 ml/min, P less than 0.001). The activity of 1-hydroxylase was by far the major factor determining serum %1,25(OH)2[3H]D3. When different acute doses of 25(OH)D3 were given to rats with identical specific activities of 1-hydroxylase, the resulting 1,25(OH)2D3 concentrations in serum correlated with the 25(OH)D3 dose (r = 0.99, P less than 0.001). We conclude that the behavior of 1-hydroxylase in vivo is analogous to the classic behavior in vitro of an enzyme functioning below its Michaelis constant (Km). The amount of 1-hydroxylase present in renal mitochondria determines the fraction (not simply the quantity) of 25(OH)D metabolized to 1,25(OH)2D3 in vivo.

The covalent binding of cyclosporin a to rat liver macromolecules in vivo and in vitro: The role of cytochrome P-450

Toxicology ◽  
1987 ◽  
Vol 47 (3) ◽  
pp. 277-284 ◽  
Author(s):  
J. Fred Nagelkerke ◽  
Roeline B. Tijidens ◽  
Erik P. Schwarz ◽  
Marjolein F.G. Winters ◽  
Leendert C. Paul ◽  
...  
Keyword(s):  
Cyclosporin A ◽  
Rat Liver ◽  
Covalent Binding ◽  
Cytochrome P 450

Evidence for the existence of a C25-sesterterpene pathway of steroidogenesis not involving cholesterol as an obligatory intermediate

1983 ◽  
Vol 61 (7) ◽  
pp. 714-721 ◽  
Author(s):  
Bhagu R. Bhavnani ◽  
Tony Wong
Keyword(s):  
Isotope Dilution ◽  
Guinea Pigs ◽  
Radiochemical Purity ◽  
Specific Activity ◽  
Dilution Technique ◽  
Isotope Dilution Technique ◽  
Alternate Pathway

Previous in vitro studies had indicated the possibility of steroidogenesis through a C25-sesterterpene pathway in which squalene and cholesterol are not required as obligatory intermediates. To investigate whether such a pathway exists in vivo, the precursor role of [7-3H]23,24-dinor-5-cholene-3β,20-diol in the in vivo formation of cortisol by the guinea pigs was studied. The [3H]23,24-dinor-5-cholene-3β,20-diol was synthesized by reacting [3H]pregnenolone acetate with a Grignard reagent. The product was purified by chromatography and its radiochemical purity was established by the isotope dilution technique. In the first experiment a total of 134 × 106 dpm of [3H]23,24-dinor-5-cholene-3β,20-diol was injected subcutaneously into three guinea pigs. Urine was collected for 8 days and was pooled. Only 12% of the administered dose was excreted in the urine. The urine was extracted and a neutral extract (3 × 106 dpm) was obtained. From this extract 2.3 mg of cortisol containing 2.9 × 104 dpm was isolated. Radiochemical purity of the isolated cortisol was established by the isotope dilution technique. Radiochemical purity was further confirmed by conversion to cortisol 21-acetate and subsequently to 11β-hydroxyandrost-4-ene-3,17-dione and recrystallization to constant specific activity. The results of this experiment were confirmed by repeating the experiment with a higher specific activity [3H]23,24-dinor-5-cholene-3β,20-diol. These results indicate that the C25-sesterterpene pathway is a possible in vivo alternate pathway of steroidogenesis, not involving either squalene or cholesterol as obligatory intermediates.

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