scholarly journals The kinetic characteristics of human and trypanosomatid phosphofructokinases for the reverse reaction

2019 ◽  
Vol 476 (2) ◽  
pp. 179-191 ◽  
Author(s):  
Peter M. Fernandes ◽  
James Kinkead ◽  
Iain W. McNae ◽  
Frédéric Bringaud ◽  
Paul A.M. Michels ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Reverse Reaction ◽  
Kinetic Properties ◽  
Kinetic Characteristics ◽  
Inorganic Pyrophosphate ◽  
Physiological Relevance ◽  
Fructose 1,6 Bisphosphate

Abstract Eukaryotic ATP-dependent phosphofructokinases (PFKs) are often considered unidirectional enzymes catalysing the transfer of a phospho moiety from ATP to fructose 6-phosphate to produce ADP and fructose 1,6-bisphosphate. The reverse reaction is not generally considered to occur under normal conditions and has never been demonstrated for any eukaryotic ATP-dependent PFKs, though it does occur in inorganic pyrophosphate-dependent PFKs and has been experimentally shown for bacterial ATP-dependent PFKs. The evidence is provided via two orthogonal assays that all three human PFK isoforms can catalyse the reverse reaction in vitro, allowing determination of kinetic properties. Additionally, the reverse reaction was shown possible for PFKs from three clinically important trypanosomatids; these enzymes are contained within glycosomes in vivo. This compartmentalisation may facilitate reversal, given the potential for trypanosomatids to have an altered ATP/ADP ratio in glycosomes compared with the cytosol. The kinetic properties of each trypanosomatid PFK were determined, including the response to natural and artificial modulators of enzyme activity. The possible physiological relevance of the reverse reaction in trypanosomatid and human PFKs is discussed.

Neutral endopeptidase 24.11 is important for the degradation of both endogenous and exogenous glucagon in anesthetized pigs

2004 ◽  
Vol 287 (3) ◽  
pp. E431-E438 ◽  
Author(s):  
Ramona Trebbien ◽  
Letty Klarskov ◽  
Mette Olesen ◽  
Jens J. Holst ◽  
Richard D. Carr ◽  
...  
Keyword(s):  
Plasma Levels ◽  
Neutral Endopeptidase ◽  
Metabolic Clearance ◽  
Clearance Rates ◽  
Important Mediator ◽  
Physiological Relevance ◽  
Glucagon Concentration

Glucagon has a short plasma t1/2in vivo, with renal extraction playing a major role in its elimination. Glucagon is degraded by neutral endopeptidase (NEP) 24.11 in vitro, but the physiological relevance of NEP 24.11 in glucagon metabolism is unknown. Therefore, the influence of candoxatril, a selective NEP inhibitor, on plasma levels of endogenous and exogenous glucagon was examined in anesthetized pigs. Candoxatril increased endogenous glucagon concentrations, from 6.3 ± 2.5 to 20.7 ± 6.3 pmol/l [COOH-terminal (C)-RIA, P < 0.05]. During glucagon infusion, candoxatril increased the t1/2determined by C-RIA (from 3.0 ± 0.5 to 17.0 ± 2.5 min, P < 0.005) and midregion (M)-RIA (2.8 ± 0.5 to 17.0 ± 3.0 min, P < 0.01) and reduced metabolic clearance rates (MCR; 19.1 ± 3.2 to 9.4 ± 2.0 ml·kg−1·min−1, P < 0.02, C-RIA; 19.2 ± 4.8 to 9.0 ± 2.3 ml·kg−1·min−1, P < 0.05, M-RIA). However, neither t1/2nor MCR determined by NH2-terminal (N)-RIA were significantly affected ( t1/2, 2.7 ± 0.4 to 4.5 ± 1.6 min; MCR, 30.3 ± 6.4 to 28.5 ± 9.0 ml·kg−1·min−1), suggesting that candoxatril had no effect on NH2-terminal degradation but leads to the accumulation of NH2-terminally truncated forms of glucagon. Determination of arteriovenous glucagon concentration differences revealed that renal glucagon extraction was reduced (but not eliminated) by candoxatril (from 40.4 ± 3.8 to 18.6 ± 4.1%, P < 0.02, C-RIA; 29.2 ± 3.1 to 14.7 ± 2.2%, P < 0.02, M-RIA; 26.5 ± 4.0 to 19.7 ± 3.5%, P < 0.06, N-RIA). Femoral extraction was reduced by candoxatril when determined by C-RIA (from 22.7 ± 2.4 to 8.0 ± 5.1%, P < 0.05) but was not changed significantly when determined using M- or N-RIAs (10.0 ± 2.8 to 4.7 ± 3.7%, M-RIA; 10.5 ± 2.5 to 7.8 ± 4.2%, N-RIA). This study provides evidence that NEP 24.11 is an important mediator of the degradation of both endogenous and exogenous glucagon in vivo.

A Molecular Approach to Immunoscintigraphy: A Study of the T-Antigen Conformation on the Surface of Tumors

1987 ◽  
Vol 26 (01) ◽  
pp. 1-6 ◽  
Author(s):  
S. Selvaraj ◽  
M. R. Suresh ◽  
G. McLean ◽  
D. Willans ◽  
C. Turner ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Tumor Cell ◽  
T Antigen ◽  
Human Carcinomas ◽  
Animal Tumor ◽  
Synthetic Antigens

The role of glycoconjugates in tumor cell differentiation has been well documented. We have examined the expression of the two anomers of the Thomsen-Friedenreich antigen on the surface of human, canine and murine tumor cell membranes both in vitro and in vivo. This has been accomplished through the synthesis of the disaccharide terminal residues in both a and ß configuration. Both entities were used to generate murine monoclonal antibodies which recognized the carbohydrate determinants. The determination of fine specificities of these antibodies was effected by means of cellular uptake, immunohistopathology and immunoscintigraphy. Examination of pathological specimens of human and canine tumor tissue indicated that the expressed antigen was in the β configuration. More than 89% of all human carcinomas tested expressed the antigen in the above anomeric form. The combination of synthetic antigens and monoclonal antibodies raised specifically against them provide us with invaluable tools for the study of tumor marker expression in humans and their respective animal tumor models.

Storage of Human Blood Platelets

1974 ◽  
Vol 32 (02/03) ◽  
pp. 405-416 ◽  
Author(s):  
M. R Hardeman ◽  
Carina J L. Heynens
Keyword(s):  
Storage Temperature ◽  
Platelet Rich Plasma ◽  
Blood Platelets ◽  
Storage Period ◽  
Serotonin Uptake ◽  
Hypotonic Shock ◽  
Glycolytic Intermediates

SummaryStorage experiments were performed at 4°, 25° and 37° C with platelet-rich plasma under sterile conditions. In some experiments also the effect of storing platelets at 4° C in whole blood was investigated.Before, during and after three days of storage, the platelets were tested at 37° C for their serotonin uptake and response to hypotonic shock. In addition some glycolytic intermediates were determined.A fair correlation was noticed between the serotonin uptake and hypotonic shock experiments. Both parameters were best maintained at 25° C. Also platelet counting, performed after the storage period, indicated 25° C as the best storage temperature. Determination of glycolytic intermediates did not justify any conclusion regarding the optimal storage temperature. Of the various anticoagulants studied, ACD and heparin gave the best results as to the serotonin uptake and hypotonic shock response, either with fresh or stored platelets. The use of EDTA resulted in the lowest activity, especially after storage.The results of these storage experiments in vitro, correspond well with those in vivo reported in the literature.

Carbon dots derived from Fusobacterium nucleatum for intracellular determination of Fe3+ and bioimaging both in vitro and in vivo

2021 ◽  
Author(s):  
Lijuan Liu ◽  
Shengting Zhang ◽  
Xiaodan Zheng ◽  
Hongmei Li ◽  
Qi Chen ◽  
...  
Keyword(s):  
Carbon Dots ◽  
Fusobacterium Nucleatum ◽  
Living Cells ◽  
First Time ◽  
Fluorescent Carbon Dots ◽  
Fluorescent Carbon

Fusobacterium nucleatum has been employed for the first time to synthesize fluorescent carbon dots which could be applied for the determination of Fe3+ ions in living cells and bioimaging in vitro and in vivo with excellent biocompatibility.

Factors affecting in vitro and in vivo antioxidant effects. Experimental conditions and nature of oxidants determine antioxidant efficacy

2021 ◽  
pp. 1-9
Author(s):  
Etsuo Niki
Keyword(s):  
Biological Molecules ◽  
Experimental Conditions ◽  
Factors Affecting ◽  
Beneficial Effects ◽  
Multiple Oxidants ◽  
Antioxidant Effects

Reactive oxygen and nitrogen species have been implicated in the onset and progression of various diseases and the role of antioxidants in the maintenance of health and prevention of diseases has received much attention. The action and effect of antioxidants have been studied extensively under different reaction conditions in multiple media. The antioxidant effects are determined by many factors. This review aims to discuss several important issues that should be considered for determination of experimental conditions and interpretation of experimental results in order to understand the beneficial effects and limit of antioxidants against detrimental oxidation of biological molecules. Emphasis was laid on cell culture experiments and effects of diversity of multiple oxidants on antioxidant efficacy.

Antimicrobial and antibiofilm activities of meropenem loaded-mesoporous silica nanoparticles against carbapenem-resistant Pseudomonas aeruginosa

2021 ◽  
pp. 088532822110038
Author(s):  
Mohammad Yousef Memar ◽  
Mina Yekani ◽  
Hadi Ghanbari ◽  
Edris Nabizadeh ◽  
Sepideh Zununi Vahed ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Mesoporous Silica ◽  
Silica Nanoparticles ◽  
Mesoporous Silica Nanoparticles ◽  
Carbapenem Resistant ◽  
Toxicity In Vitro ◽  
Different Levels

The aims of the present study were the determination of antimicrobial and antibiofilm effects of meropenem-loaded mesoporous silica nanoparticles (MSNs) on carbapenem resistant Pseudomonas aeruginosa ( P. aeruginosa) and cytotoxicity properties in vitro. The meropenem-loaded MSNs had shown antibacterial and biofilm inhibitory activities on all isolates at different levels lower than MICs and BICs of meropenem. The viability of HC-04 cells treated with serial concentrations as MICs and BICs of meropenem-loaded MSNs was 92–100%. According to the obtained results, meropenem-loaded MSNs display the significant antibacterial and antibiofilm effects against carbapenem resistant and biofilm forming P. aeruginosa and low cell toxicity in vitro. Then, the prepared system can be an appropriate option for the delivery of carbapenem for further evaluation in vivo assays.

scholarly journals Cofactor bypass variants reveal a conformational control mechanism governing cell wall polymerase activity

2016 ◽  
Vol 113 (17) ◽  
pp. 4788-4793 ◽  
Author(s):  
Monica Markovski ◽  
Jessica L. Bohrhunter ◽  
Tania J. Lupoli ◽  
Tsuyoshi Uehara ◽  
Suzanne Walker ◽  
...  
Keyword(s):  
Polymerase Activity ◽  
Activation Mechanism ◽  
Phenotypic Analysis ◽  
Outer Membranes ◽  
Division Site ◽  
Physiological Relevance ◽  
Membrane Lipoproteins ◽  
Shed Light

To fortify their cytoplasmic membrane and protect it from osmotic rupture, most bacteria surround themselves with a peptidoglycan (PG) exoskeleton synthesized by the penicillin-binding proteins (PBPs). As their name implies, these proteins are the targets of penicillin and related antibiotics. We and others have shown that the PG synthases PBP1b and PBP1a ofEscherichia colirequire the outer membrane lipoproteins LpoA and LpoB, respectively, for their in vivo function. Although it has been demonstrated that LpoB activates the PG polymerization activity of PBP1b in vitro, the mechanism of activation and its physiological relevance have remained unclear. We therefore selected for variants of PBP1b (PBP1b*) that bypass the LpoB requirement for in vivo function, reasoning that they would shed light on LpoB function and its activation mechanism. Several of these PBP1b variants were isolated and displayed elevated polymerization activity in vitro, indicating that the activation of glycan polymer growth is indeed one of the relevant functions of LpoB in vivo. Moreover, the location of amino acid substitutions causing the bypass phenotype on the PBP1b structure support a model in which polymerization activation proceeds via the induction of a conformational change in PBP1b initiated by LpoB binding to its UB2H domain, followed by its transmission to the glycosyl transferase active site. Finally, phenotypic analysis of strains carrying a PBP1b* variant revealed that the PBP1b–LpoB complex is most likely not providing an important physical link between the inner and outer membranes at the division site, as has been previously proposed.

scholarly journals Cortistatin Is Not a Somatostatin Analogue but Stimulates Prolactin Release and Inhibits GH and ACTH in a Gender-Dependent Fashion: Potential Role of Ghrelin

Endocrinology ◽  
2011 ◽  
Vol 152 (12) ◽  
pp. 4800-4812 ◽  
Author(s):  
José Córdoba-Chacón ◽  
Manuel D. Gahete ◽  
Ana I. Pozo-Salas ◽  
Antonio J. Martínez-Fuentes ◽  
Luis de Lecea ◽  
...  
Keyword(s):  
Metabolic Phenotype ◽  
Somatostatin Analogue ◽  
Growth Hormone Secretagogue Receptor ◽  
Growth Hormone Secretagogue ◽  
Ancestral Gene ◽  
Ghrelin Receptor ◽  
Physiological Relevance

Cortistatin (CST) and somatostatin (SST) evolve from a common ancestral gene and share remarkable structural, pharmacological, and functional homologies. Although CST has been considered as a natural SST-analogue acting through their shared receptors (SST receptors 1–5), emerging evidence indicates that these peptides might in fact exert unique roles via selective receptors [e.g. CST, not SST, binds ghrelin receptor growth hormone secretagogue receptor type 1a (GHS-R1a)]. To determine whether the role of endogenous CST is different from SST, we characterized the endocrine-metabolic phenotype of male/female CST null mice (cort−/−) at hypothalamic-pituitary-systemic (pancreas-stomach-adrenal-liver) levels. Also, CST effects on hormone expression/secretion were evaluated in primary pituitary cell cultures from male/female mice and female primates (baboons). Specifically, CST exerted an unexpected stimulatory role on prolactin (PRL) secretion, because both male/female cort−/− mice had reduced PRL levels, and CST treatment (in vivo and in vitro) increased PRL secretion, which could be blocked by a GHS-R1a antagonist in vitro and likely relates to the decreased success of female cort−/− in first-litter pup care at weaning. In contrast, CST inhibited GH and adrenocorticotropin-hormone axes in a gender-dependent fashion. In addition, a rise in acylated ghrelin levels was observed in female cort−/− mice, which were associated with an increase in stomach ghrelin/ghrelin O-acyl transferase expression. Finally, CST deficit uncovered a gender-dependent role of this peptide in the regulation of glucose-insulin homeostasis, because male, but not female, cort−/− mice developed insulin resistance. The fact that these actions are not mimicked by SST and are strongly gender dependent offers new grounds to investigate the hitherto underestimated physiological relevance of CST in the regulation of physiological/metabolic processes.

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