scholarly journals Human cystathionine γ-lyase: developmental and in vitro expression of two isoforms

2000 ◽  
Vol 347 (1) ◽  
pp. 291-295 ◽  
Author(s):  
Anna-Liisa LEVONEN ◽  
Risto LAPATTO ◽  
Mika SAKSELA ◽  
Kari O. RAIVIO
Keyword(s):  
Human Liver ◽  
Human Kidney ◽  
Enzymic Activity ◽  
Developmental Changes ◽  
Transcriptional Level ◽  
Mrna Isoforms ◽  
Gestational Week ◽  
Neonatal Liver ◽  
Increased Activity

Cystathionine γ-lyase (CGL) is the last enzyme of the trans-sulphuration pathway, which converts methionine into cysteine. To study the possible differences in enzymic activity of the two human cystathionine γ-lyase isoforms characterized earlier, these were separately expressed in human kidney embryonic 293T cells. Furthermore, developmental changes in the expression of the two mRNA forms as well as the enzymic activity in human liver were studied, as it has been postulated that a change in the relative expression of CGL isoforms causes the postnatal increase in CGL activity. Transfection with the longer isoform increased the CGL activity 1.5-fold, while the activity of the cells transfected with the shorter form did not differ from the basal activity. In human liver samples, CGL activity was only detected in adult tissue (68±9 nmol of cysteine/h per mg of protein), whereas activity in fetal, premature and full-term neonatal liver tissue was undetectable. In contrast, strong mRNA expression of both mRNA isoforms was detected from the 19th gestational week onwards and the longer form of CGL appeared to be predominant. The expression of the two mRNA forms varied in parallel. In conclusion, we have shown that only cells overexpressing the longer form of CGL have increased activity, and CGL appears to be regulated at the post-transcriptional level during development.

scholarly journals Cytokine induction of haem oxygenase mRNA in mouse liver. Interleukin 1 transcriptionally activates the haem oxygenase gene

1993 ◽  
Vol 290 (2) ◽  
pp. 343-347 ◽  
Author(s):  
M Rizzardini ◽  
M Terao ◽  
F Falciani ◽  
L Cantoni
Keyword(s):  
Interleukin 1 ◽  
Interleukin 2 ◽  
Northern Blotting ◽  
Enzymic Activity ◽  
Transcriptional Level ◽  
Protective Mechanisms ◽  
Biological Responses ◽  
Haem Oxygenase

Accumulation of the mRNA coding for haem oxygenase (HO, EC 1.14.99.3) was stimulated by treating mice with endotoxin (lipopolysaccharide, LPS; 20 micrograms/mouse intraperitoneally), suggesting that haem catabolism is a target of infection and inflammation in vivo. Therefore various cytokines, possible mediators for the biological responses to LPS, were administered intraperitoneally to mice, and the levels of HO mRNA were measured by Northern-blotting analysis using the rat HO cDNA as a probe [Shibahara, Müller, Taguchi and Yoshida (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7865-7869]. Marked induction of HO mRNA was observed 2 h after administration of interleukin 1 (IL-1) (34-fold) and tumour necrosis factor (19.5-fold) (5 micrograms/mouse), whereas interleukin 6 (6.2 micrograms/mouse) was much less active (3.5-fold) and interleukin 2 (25 micrograms/mouse) and interferon-gamma (3 micrograms/mouse) were ineffective. HO mRNA induced by the cytokines of LPS accumulated rapidly (maximum at 1-2 h after administration), preceding the elevation of HO enzymic activity. Treatment of mice with IL-1 stimulated the transcription of the HO gene by 4-fold, as assessed by in vitro nuclear-run-on assay. These results indicate that enzymic haem catabolism in the liver is a process inducible in vivo by inflammatory cytokines, which up-regulate HO synthesis at the transcriptional level. Increased removal of haem might be part of the protective mechanisms elicited by the acute-phase response, possibly to reduce the pro-oxidant state of the cell.

scholarly journals Glycosylation of ribonuclease A catalysed by rabbit liver extracts

1975 ◽  
Vol 146 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Z Khalkhall ◽  
R D Marshall
Keyword(s):  
Metal Ion ◽  
Ribonuclease A ◽  
Enzymic Activity ◽  
Rabbit Liver ◽  
Absolute Requirement ◽  
Pancreatic Ribonuclease A ◽  
Triton X ◽  
Increased Activity ◽  
Asparagine Residue

Crude extracts of rabbit liver catalyse in vitro the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to bovine pancreatic ribonuclease A. The enzymic activity is contained in rough endoplasmic reticulum. It has an absolute requirement for a bivalent metal ion: Co-2+ greater than Mn-2+ greater than Ni-2+. Mg-2+ is ineffective. There is enzymic activity in the absence of detergent, but increased activity is observed in the presence of Triton X-100. The site of glycosylation of ribonuclease A is asparagine-34, and glycosylation occurs only at this point. These findings agree with the hypothesis that the sequence Asn-X-Thr(Ser) (where X may be one of a number of types of amino acid) is a necessary, but not sufficient, condition for N-acetylglucosaminylation of a protein-bound asparagine residue.

scholarly journals Uptake of iodinated human kidney α-D-mannosidase by rat liver- Association with membrane elements and stability in vivo and in vitro

1976 ◽  
Vol 159 (3) ◽  
pp. 729-736 ◽  
Author(s):  
D V Marinković ◽  
S L Petrović ◽  
J V Martinović ◽  
J N Marinković
Keyword(s):  
Plasma Membranes ◽  
Specific Radioactivity ◽  
Human Kidney ◽  
Particulate Material ◽  
Enzymic Activity ◽  
Free Form ◽  
Rat Tissues ◽  
Liver And Kidney

1. Human kidney α-D-mannosidase (form A) was labelled with 125I to a specific radio-activity of approx. 2250muCi/mg of protein, essentially without loss of enzymic activity. The enzymic activity and radioactivity of the iodinated material also co-migrated in gel filtration and gel electrophoresis. 2. The binding of 125I-labelled mannosidase in vitro to particulate material in liver and kidney homogenates was of the other of 2 pg/mg of particulate material in liver and kidney homogenates was of the order of 2pg/mg of particulate protein withing 16h at 37 degrees C, and essentially zero in intervals of up to 60 min. The degradation in vitro of labelled exogenous mannosidase was of the order of 10-20pg/ 16th per mg of protein in postnuclear supernatant, and it was saturated entirely within 1h at 37 degrees C. 3. The binding of labelled mannosidase in vivo to particulate elements of liver homogenates 60 min after intravenous injection was at least 10 times higher in terms of specific radioactivity than the highest value attainable in vitro. Virtually all exogenous enzyme bound to liver particulate material could be recovered in macromolecular form after disruption of membranes by detergents. 4. The radioactive enzyme bound to liver particulate material could be detached almost completely by shearing, repeated freezing and thawing, and exposure to strong detergents under conditions that do not eliminate rough-endoplasmic-membrane structure. It could bot be released, however, by high salt concentration (0.5M-KC1) or by exposure to weak detergents such as Tween 80. The particle-bound enzyme should thus be associated with plasma membranes and lysosome-like elements. 5. Of the rat tissues studied, only liver could approach, within 60 min after the injection, the concentration of exogenous mannosidase found in the blood serum. The activity per g tissue weight fell progressively from liver (60% of serum value) to kidney (16% of serum value), lung (8% of serum vlaue), spleen (6% of serum value) and brain (0.9% of serum value). Most of the radioactive enzyme found in tissues other than liver appeared to be present in a free form, whereas in liver more than 50% of the labelled enzyme was associated with membrane elements.

Characterizations of Human UDP-Glucuronosyltransferase Enzymes in the Conjugation of p-Cresol

2020 ◽  
Vol 176 (2) ◽  
pp. 285-296 ◽  
Author(s):  
Yan Rong ◽  
Tony K L Kiang
Keyword(s):  
Human Liver ◽  
Substrate Inhibition ◽  
Liver Microsomes ◽  
Human Kidney ◽  
Human Liver Microsomes ◽  
Uremic Toxin ◽  
Inverse Association ◽  
Kidney Microsomes ◽  
Udp Glucuronosyltransferase

Abstract p-Cresol is a uremic toxin that is formed by intestinal microbiota and extensively conjugated by first-pass metabolism. p-Cresol glucuronide exerts various forms of cellular toxicity in vitro and is accumulated in the plasma of subjects with kidney disease, where associations with adverse cardiovascular and renal outcomes are evident. The objective of this study was to determine the contributions of human UDP-glucuronosyltransferase (UGT) enzymes in the formation of p-cresol glucuronide. Utilizing commonly expressed hepatic or renal human recombinant UGTs (ie, hrUGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B15, and 2B17), hrUGT1A6 and hrUGT1A9 exhibited the highest catalytic activities in the generation of p-cresol glucuronide. The kinetics of p-cresol glucuronide formation in hrUGT1A6 and pooled human liver microsomes were best described by the Hill equation and in hrUGT1A9 and pooled human kidney microsomes by substrate inhibition. Using inhibitory and selective UGT inhibitors (ie, acetaminophen or amentoflavone for UGT1A6 and niflumic acid for UGT1A9), UGT1A6 was identified the predominant enzyme responsible for p-cresol glucuronide production in pooled human liver (78.4%–81.3% contribution) and kidney (54.3%–62.9%) microsomes, whereas UGT1A9 provided minor contributions (2.8% and 35.5%, respectively). The relative contributions of UGT1A6 (72.6 ± 11.3%, mean ± SD) and UGT1A9 (5.7 ± 4.1%) in individual human liver microsomes from 12 adult donors were highly variable, where an inverse association (R = −.784, p = .003) between UGT1A6 contribution and UGT1A9 probe substrate activity (ie, mycophenolic acid) was evident. Our novel findings provide valuable tools for conducting further mechanistic studies and for designing clinical interventions to mitigate the toxicities associated with p-cresol glucuronide.

Human liver cell cultivation for long-term in vitro toxicity studies in a miniaturized multi-compartment 3D bioreactor system

2011 ◽  
Vol 49 (01) ◽  
Author(s):  
SA Hoffmann ◽  
M Lübberstedt ◽  
U Müller-Vieira ◽  
D Knobeloch ◽  
A Nüssler ◽  
...  
Keyword(s):  
Liver Cell ◽  
Human Liver ◽  
In Vitro Toxicity ◽  
Cell Cultivation ◽  
Toxicity Studies ◽  
Human Liver Cell ◽  
Bioreactor System

Binding of Tissue Plasminogen Activator to Vascular Grafts

1989 ◽  
Vol 61 (01) ◽  
pp. 131-136 ◽  
Author(s):  
Richard A Harvey ◽  
Hugh C Kim ◽  
Jonathan Pincus ◽  
Stanley Z Trooskin ◽  
Josiah N Wilcox ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Polymer Surface ◽  
Enzymic Activity ◽  
Vascular Prostheses ◽  
Chemically Modified ◽  
Insoluble Surfactant ◽  
Tissue Plasminogen

SummaryTissue plasminogen activator labeled with radioactive iodine (125I-tPA) was immobilized on vascular prostheses chemically modified with a thin coating of water-insoluble surfactant, tridodecylmethylammonium chloride (TDM AC). Surfactant- treated Dacron, polytetrafluoroethylene (PTFE), silastic, polyethylene and polyurethane bound appreciable amounts of 125I- tPA (5-30 μg 125I-tPA/cm2). Upon exposure to human plasma, the amount of 125I-tPA bound to the surface shows an initial drop during the first hour of incubation, followed by a slower, roughly exponential release with a t½ of appoximately 75 hours. Prostheses containing bound tPA show fibrinolytic activity as measured both by lysis of clots formed in vitro, and by hydrolysis of a synthetic polypeptide substrate. Prior to incubation in plasma, tPA bound to a polymer surface has an enzymic activity similar, if not identical to that of the native enzyme in buffered solution. However, exposure to plasma causes a decrease in the fibrinolytic activity of both bound tPA and enzyme released from the surface of the polymer. These data demonstrate that surfactant-treated prostheses can bind tPA, and that these chemically modified devices can act as a slow-release drug delivery system with the potential for reducing prosthesis-induced thromboembolism.

Triploidy in a Live-Born Extremely Low Birth Weight Twin: Clinical Aspects

2020 ◽  
Author(s):  
Liliya Vakrilova ◽  
Stanislava Hitrova-Nikolova ◽  
Irena Bradinova
Keyword(s):  
Left Lung ◽  
Extremely Low Birth Weight ◽  
Clinical Aspects ◽  
Severe Respiratory Distress ◽  
Vitro Fertilization ◽  
Gestational Week ◽  
Polymerase Chain ◽  
Fluorescent Polymerase Chain Reaction ◽  
Second Twin

AbstractTriploidy is a rare chromosomal aberration characterized by a karyotype with 69 chromosomes. Triploid fetuses usually are miscarried in early pregnancy. We present a case of a triploid twin and a genetically unaffected co-twin, conceived through in vitro fertilization. A discordant growth was registered at 20 weeks of gestation. Cesarean section was performed at 355/7 gestational week. The second twin was extremely growth restricted female (780 g) with oligohydramnios and severe respiratory distress, and died at 20 hours of age. The autopsy revealed unilobar left lung, bilobar right lung, and cysts of the terminal bronchioles. Quantitative fluorescent polymerase chain reaction detected triploidy compatible pattern. So, early intrauterine growth restriction may be a sign of triploidy, which must be proven by pre or postnatal genetic testing.

In Vitro Study of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (RDX) Metabolism in Human Liver

2008 ◽  
Author(s):  
Cheng J. Cao ◽  
Gunda Reddy ◽  
Desmond I. Bannon ◽  
Mark S. Johnson
Keyword(s):  
Human Liver ◽  
In Vitro Study ◽  
Vitro Study

AAnti-Inflammatory Effects of Plasma Circulating Exosomes Obtained from Normal-Weight and Obese Subjects on Hepatocytes

2020 ◽  
Vol 20 ◽  
Author(s):  
Reza Afrisham ◽  
Sahar Sadegh-Nejadi ◽  
Reza Meshkani ◽  
Solaleh Emamgholipour ◽  
Molood Bagherieh ◽  
...  
Keyword(s):  
Inflammatory Cytokines ◽  
Hepg2 Cells ◽  
Human Liver ◽  
Liver Cells ◽  
Normal Weight ◽  
Protein Levels ◽  
Human Liver Cells ◽  
Tnf Α ◽  
Anti Inflammatory

Introduction: Obesity is a disorder with low-grade chronic inflammation that plays a key role in the hepatic inflammation and steatosis. Moreover, there are studies to support the role of exosomes in the cellular communications, the regulation of metabolic homeostasis and immunomodulatory activity. Accordingly, we aimed to evaluate the influence of plasma circulating exosomes derived from females with normal-weight and obesity on the secretion of inflammatory cytokines in human liver cells. Methods: Plasma circulating exosomes were isolated from four normal (N-Exo) and four obese (O-Exo) women. The exosomes were characterized and approved for CD63 expression (common exosomal protein marker) and morphology/size using the western blot and TEM methods, respectively. The exosomes were used for stimulation of HepG2 cells in vitro. After 24 h incubation, the protein levels of TNF-α,IL-6, and IL-1β were measured in the culture supernatant of HepG2 cells using the ELISA kit. Results: The protein levels of IL-6 and TNF-α in the cells treated with O-Exo and N-Exo reduced significantly in comparison with control group (P=0.039 and P<0.001 respectively), while significance differences were not found between normal and obese groups (P=0.808, and P=0.978 respectively). However, no significant differences were found between three groups in term of IL-1β levels (P=0.069). Based on the correlation analysis, the protein levels of IL-6 were positively correlated with TNF-α (r 0.978, P<0.001). Conclusion: These findings suggest that plasma circulating exosomes have probably anti-inflammatory properties independently from body mass index and may decrease the secretion of inflammatory cytokines in liver. However, further investigations in vitro and in vivo are needed to address the anti-inflammatory function of N-Exo and O-Exo in human liver cells and/or other cells.

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