HUMAN HEPATOCYTES CONTAIN HIGH MOLECULAR WEIGHT POLYPEPTIDES OF FACTOR VIII

1987 ◽  
Author(s):  
J Ingerslev ◽  
B Sloth Christiansen ◽  
A Bukh ◽  
S Stenbjerg ◽  
T Munck Jørgensen ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Polyclonal Antibodies ◽  
Plasma Source ◽  
Isolated Hepatocytes ◽  
Human Hepatocytes ◽  
Isolated Cells ◽  
Left Liver Lobe ◽  
Viable Cells ◽  
A Cell

Human hepatocytes were isolated by the two-step collagenase technique applied on distal left liver lobe. Homogenous and large cells were isolated revealing hepatocyte characteristics by light-microscopy. Hepatocytes were washed repeatedly in albumine buffer (5%), resuspended in the same buffer and sonicated using a cell density of 0.75 × 106 cells/ml. In some cases cells were separated from non-viable cells by flotation on a linear Percoll gradient. Supernate material after sonication was subjected to ELISA for VIII:Ag using human antibodies and vWf:Ag by polyclonal antibodies. Freshly isolated cells contained at least 0.25 IU/ 0.75 × 106 hepatocytes, whereas the vWf:Ag was below 0.01 IU/ 0.75 × 106 cells. The material obtained from sonication was further studied using fast protein liquid chromatography by Mono-Q HR 5/5 revealing a single peak of VIII: Ag eluting in the same position as the high molecular weight polypeptides of VIII :Ag of high purity FVIII derived from the plasma source. Isolated hepatocytes also were cultivated at 37°C in medium RPMI 1640 supplemented with Ultroser G (4%), glutamine and antibiotics. Cells secreted increasing quantities of albumin, fitrinogpn and protease-inhibitors. The supernatants also contained VIII: Ag in quantities ranging from 0.04 - 0.17 IU/ml after 24 hours, but no further secretion was observed. No vWf: Ag could be detected. Cells harvested and sonicated after 30 hours of culture only contained 0.04 IU/ 0.75 × 106 cells. Our results shows, that VIII :Ag is present in freshly isolated human hepatocytes and that only traces of vWf:Ag is found. A hepatocyte site of production of VIII is speculated. These very preliminary findings do not permit conclusions concerning active synthesis of VIII in hepatocytes. Further studies are underway.

scholarly journals Characterization of a Novel Fructosyltransferase from Lactobacillus reuteri That Synthesizes High-Molecular-Weight Inulin and Inulin Oligosaccharides

2002 ◽  
Vol 68 (9) ◽  
pp. 4390-4398 ◽  
Author(s):  
S. A. F. T. van Hijum ◽  
G. H. van Geel-Schutten ◽  
H. Rahaoui ◽  
M. J. E. C. van der Maarel ◽  
L. Dijkhuizen
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Lactobacillus Reuteri ◽  
Bacterial Strains ◽  
Potential Health ◽  
Isolation And Characterization ◽  
A Cell ◽  
Encoding Gene ◽  
First Time

ABSTRACT Fructosyltransferase (FTF) enzymes produce fructose polymers (fructans) from sucrose. Here, we report the isolation and characterization of an FTF-encoding gene from Lactobacillus reuteri strain 121. A C-terminally truncated version of the ftf gene was successfully expressed in Escherichia coli. When incubated with sucrose, the purified recombinant FTF enzyme produced large amounts of fructo-oligosaccharides (FOS) with β-(2→1)-linked fructosyl units, plus a high-molecular-weight fructan polymer (>107) with β-(2→1) linkages (an inulin). FOS, but not inulin, was found in supernatants of L. reuteri strain 121 cultures grown on medium containing sucrose. Bacterial inulin production has been reported for only Streptococcus mutans strains. FOS production has been reported for a few bacterial strains. This paper reports the first-time isolation and molecular characterization of (i) a Lactobacillus ftf gene, (ii) an inulosucrase associated with a generally regarded as safe bacterium, (iii) an FTF enzyme synthesizing both a high molecular weight inulin and FOS, and (iv) an FTF protein containing a cell wall-anchoring LPXTG motif. The biological relevance and potential health benefits of an inulosucrase associated with an L. reuteri strain remain to be established.

scholarly journals High Molecular Weight Kininogen Peptides Inhibit the Formation of Kallikrein on Endothelial Cell Surfaces and Subsequent Urokinase-Dependent Plasmin Formation

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 690-697 ◽  
Author(s):  
Yingzhang Lin ◽  
Robert B. Harris ◽  
Wuyi Yan ◽  
Keith R. McCrae ◽  
Hong Zhang ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Structural Information ◽  
Umbilical Vein ◽  
High Molecular Weight Kininogen ◽  
Cell Surfaces ◽  
Free System ◽  
Order Of Magnitude ◽  
A Cell ◽  
Umbilical Vein Endothelial Cells

A sequence of 31 amino acids (S565-K595) in domain 6 of the light chain of high molecular weight kininogen (HK) has previously been shown to be responsible for the binding of plasma prekallikrein (PK) or kallikrein. To find effective peptides that might block binding between HK and PK on cell surfaces, a new series of synthetic peptides has now been prepared that incorporates portions of this binding domain sequence. For mapping the minimal sequence within HK, these new peptides were tested for their ability to compete with HK for binding PK in a cell-free system and on human umbilical vein endothelial cells (HUVEC). In the former, at pH 7.4, the kds for binding between kallikrein and either D567-K595, S565-P594, D567-S593, or D567-T591 were all similar to that for the binding of S565-K595 (0.2 to 0.4 μmol/L), but those for the binding of D568-K595, W569-K595, and D567-P589 were an order of magnitude greater (kd = 2 to 5 μmol/L). D567-S586, the shortest chain length of the N- and C-terminal truncation sequences tested, does not effectively compete with kininogen for kallikrein binding (kd = 100 μmol/L). These results imply that D567-T591, a 25-residue peptide (HK25c), contains sufficient structural information for binding kallikrein in solution. D567-T591 also is the minimum structural sequence to block binding of kallikrein to HUVEC-bound HK (IC50 = 50 nmol/L) and to inhibit PK activation to kallikrein on the cell surface (IC50 = 80 nmol/L). In addition, D567-T591 also inhibits the generation of kallikrein-activated urokinase, which activates plasminogen to plasmin (IC50 = 100 nmol/L). Thus, HK-derived peptides may be useful compounds for modulating excessive fibrinolysis and hypotension in sepsis and multiple trauma.

The Role of Urokinase Receptor (uPAR) In Efferocytosis: uPAR Engulfs Apoptotic Cells via a Phosphatidylserine Pathway Mediated by Plasma High Molecular Weight Kininogen

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 929-929 ◽  
Author(s):  
Aizhen Yang ◽  
Jihong Dai ◽  
Raymond B. Birge ◽  
Yi Wu
Keyword(s):  
Molecular Weight ◽  
Flow Cytometry ◽  
Cell Surface ◽  
High Molecular Weight ◽  
Apoptotic Cell ◽  
Annexin V ◽  
Apoptotic Cells ◽  
High Molecular Weight Kininogen ◽  
Viable Cells

Abstract Abstract 929 Phagocytosis of apoptotic cells by phagocytes, also known as efferocytosis, is essential for maintaining normal tissue homeostasis and regulating immune responses. Defects in rapid clearance of apoptotic cells lead to the release of immunogenic cellular contents, which may cause tissue damage and autoimmune disease. Phagocytic receptors differentiate apoptotic cells from viable cells by recognizing ‘don't eat- or eat-me’ signals on the cell surface. Recently, we and others have reported the role of uPAR in mediating efferocytosis. In this study, we examined the mechanism by which uPAR recognizes and internalizes apoptotic cells. By flow cytometry-based in vivo and in vitro phagocytosis assay, we found that in knockout mice the lack of uPAR expression on macrophages decreased their apoptotic cell engulfing activity by >35%. Conversely, soluble uPAR and polyclonal anti-uPAR antibodies (Ab) suppressed the internalization of apoptotic cells by macrophages. However, there was no defect in uPAR-/- macrophage uptake of viable cells, suggesting that uPAR plays a specific role in phagocytosis of apoptotic cells. We established a HEK 293 cell line expressing human full-length uPAR (293-uPAR). In these cells, uPAR-mediated phagocytosis of apoptotic cells was completely blocked by annexin V in the presence of calcium. The effect of annexin V was not observed in the absence of calcium, indicating that uPAR internalizes apoptotic cells through a phosphatidylserine pathway. We also found that uPAR-mediated uptake of apoptotic cells was completely prevented under serum-free conditions. To identify plasma proteins that may opsonize the uPAR function, we used immunodepletion method to test three known uPAR-binding proteins, vitronectin, uPA and high molecular weight kininogen (HK). Depletion of HK from serum by a polyclonal anti-HK Ab significantly reduced the engulfment of apoptotic cells by either macrophages or 293-uPAR cells in a co-culture system. In contrast, depletion of vitronectin or uPA from serum had little effect. uPAR is a GPI-anchored protein. Upon sucrose gradient ultracentrifugation, the majority of uPAR molecules were co-localized with membrane-bound HK in lipid rafts. The binding capacity of HK to apoptotic cell surface was further analyzed by flow cytometry. Phycoerythrin-labeled HK bound to apoptotic cells in a concentration-dependent manner, saturating at 300 nM. In contrast, HK did not bind to viable cells at concentrations up to 1200 nM. It is known that HK is a key component of the plasma contact system and that apoptotic cells potentiate factor Xa formation. Our new findings of the uPAR-HK-phosphatidylserine axis in efferocytosis suggest that this pathway may modulate the coagulation cascade on the surface of apoptotic cells. This pathway may also play a role in the pathogenesis of autoimmune and thrombotic disease. Disclosures: No relevant conflicts of interest to declare.

Human Liver Slices Express the Same Lidocaine Biotransformation Rate as Isolated Human Hepatocytes

1993 ◽  
Vol 21 (4) ◽  
pp. 466-469
Author(s):  
Peter Olinga ◽  
Marjolijn T. Merema ◽  
Dick K.F. Meijer ◽  
Maarten J.H. Slooff ◽  
Geny M.M. Groothuis
Keyword(s):  
Cytochrome P450 ◽  
Individual Differences ◽  
Human Liver ◽  
Isolated Hepatocytes ◽  
Human Hepatocytes ◽  
Isolated Cells ◽  
Liver Slices ◽  
Human Livers ◽  
Optimal Incubation ◽  
Human Liver Slices

In order to investigate whether liver slices are a valuable tool for the assessment of drug metabolism in human liver, we compared the phase I metabolism of lidocaine in human liver slices and hepatocytes prepared from three human livers. Lidocaine is mainly metabolised to monoethylglycinexykdide (MEGX) via a cytochrome P450-mediated N-deethylation. The results indicate that the three livers showed considerable inter-individual differences in the rate of formation of MEGX, and that this difference was equally reflected in slices and isolated cells. The use of liver slices is still under development, and optimal incubation conditions still need to be assessed. However, these results suggest that, in slices of 200–300μm thickness, virtually all hepatocytes are involved in the biotransformation of lidocaine, and that the metabolic activity is preserved equally well as in isolated hepatocytes.

scholarly journals Surface Treatment of Ultra-High Molecular Weight Polyethylene (UHMWPE) by Cold Atmospheric Plasma (CAP) for Biocompatibility Enhancement

2021 ◽  
Vol 11 (4) ◽  
pp. 1703
Author(s):  
Jack Turicek ◽  
Nicole Ratts ◽  
Matey Kaltchev ◽  
Nazieh Masoud
Keyword(s):  
Molecular Weight ◽  
Contact Angle ◽  
High Molecular Weight ◽  
Water Contact Angle ◽  
Plasma Source ◽  
Atmospheric Plasma ◽  
Water Contact ◽  
Artificial Joints ◽  
Joint Replacements ◽  
Ultra High Molecular Weight

Ultra-high molecular weight polyethylene (UHMWPE) is one of the most commonly used polymers in joint replacements because of its biologically inert properties and low friction coefficient. However, it has downfalls relating to its wear, adhesion, and lubrication. In this study, UHMWPE samples were treated with a tubular helium cold atmospheric pressure (CAP) plasma source in order to improve three properties of the polymer: (1) its wear resistance, which was characterized by durometer hardness, (2) its lubrication characterized by water contact angle, and (3) its adhesion characterized by both root mean square surface roughness (Rq) and water contact angle. The polymer was treated by two different parts of the plasma plume (the base and the tip) at two different helium flow rates (1 L/min and 2.5 L/min), for different treatment times. Results of the plasma treatment showed a decrease in the contact angle of between 32 and 54 degrees, a significant increase in the roughness by up to 10 times the pristine surface, and no substantial change in the hardness. These improvements to the adhesion and lubrication properties of the polymer examined suggest that the treated surface could be more suitable for use in artificial joints.

scholarly journals High Molecular Weight Kininogen Peptides Inhibit the Formation of Kallikrein on Endothelial Cell Surfaces and Subsequent Urokinase-Dependent Plasmin Formation

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 690-697 ◽  
Author(s):  
Yingzhang Lin ◽  
Robert B. Harris ◽  
Wuyi Yan ◽  
Keith R. McCrae ◽  
Hong Zhang ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Structural Information ◽  
Umbilical Vein ◽  
High Molecular Weight Kininogen ◽  
Cell Surfaces ◽  
Free System ◽  
Order Of Magnitude ◽  
A Cell ◽  
Umbilical Vein Endothelial Cells

Abstract A sequence of 31 amino acids (S565-K595) in domain 6 of the light chain of high molecular weight kininogen (HK) has previously been shown to be responsible for the binding of plasma prekallikrein (PK) or kallikrein. To find effective peptides that might block binding between HK and PK on cell surfaces, a new series of synthetic peptides has now been prepared that incorporates portions of this binding domain sequence. For mapping the minimal sequence within HK, these new peptides were tested for their ability to compete with HK for binding PK in a cell-free system and on human umbilical vein endothelial cells (HUVEC). In the former, at pH 7.4, the kds for binding between kallikrein and either D567-K595, S565-P594, D567-S593, or D567-T591 were all similar to that for the binding of S565-K595 (0.2 to 0.4 μmol/L), but those for the binding of D568-K595, W569-K595, and D567-P589 were an order of magnitude greater (kd = 2 to 5 μmol/L). D567-S586, the shortest chain length of the N- and C-terminal truncation sequences tested, does not effectively compete with kininogen for kallikrein binding (kd = 100 μmol/L). These results imply that D567-T591, a 25-residue peptide (HK25c), contains sufficient structural information for binding kallikrein in solution. D567-T591 also is the minimum structural sequence to block binding of kallikrein to HUVEC-bound HK (IC50 = 50 nmol/L) and to inhibit PK activation to kallikrein on the cell surface (IC50 = 80 nmol/L). In addition, D567-T591 also inhibits the generation of kallikrein-activated urokinase, which activates plasminogen to plasmin (IC50 = 100 nmol/L). Thus, HK-derived peptides may be useful compounds for modulating excessive fibrinolysis and hypotension in sepsis and multiple trauma.

Identification of high molecular weight peptides in bovine adrenomedullary chromaffin granules using monoclonal antibodies to a preproenkephalin A fusion peptide

1989 ◽  
Vol 2 (2) ◽  
pp. 119-129 ◽  
Author(s):  
S. Jackson ◽  
B. A. Spruce ◽  
D. M. Glover ◽  
B. P. Glynn ◽  
P. J. Lowry
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
High Molecular Weight ◽  
High Performance ◽  
Polyclonal Antibodies ◽  
Immunoblot Analysis ◽  
Fusion Peptide ◽  
Chromaffin Granules ◽  
Immunoreactive Band ◽  
Adrenal Chromaffin

ABSTRACT Two mouse monoclonal antibodies (PE-1 and PE-2) raised to a β-galactosidase—preproenkephalin A(69– 207) fusion peptide recognize pro-enkephalin A (pro-enk-A) peptides of 33–5 kDa isolated from bovine adrenal chromaffin granules. The preliminary characterization of the high molecular weight adrenomedullary pro-enk-A peptides recognized by PE-1 and PE-2 is described. The high molecular weight peptides were resolved after Sephadex G-50 chromatography and high-performance liquid chromatography (HPLC) into three components (peaks I, II and III). Immunoblot analysis showed each HPLC peak to be heterogeneous. Peak I contained PE-1-and PE-2-immunoreactive peptides of 33, 29, 24 and 22 kDa; peak II contained a peptide of 22 kDa recognized by PE-2, and peptides of 24 and 22 kDa recognized by PE-1; peak III contained a PE-2-immunoreactive peptide of 15 kDa and PE-1-immunoreactive peptide of 18 kDa. Using polyclonal antibodies to peptide F and methionineenkephalin-Arg6-Gly7-Leu8 (MetEnk-RGL), the 22 kDa band cross-reacted with both MetEnk-RGL and peptide F antibodies, whilst the 24 kDa band was shown to possess predominantly MetEnk-RGL immunoreactivity. The 15 kDa (PE-2-immunoreactive) band was recognized by the peptide F but not the MetEnk-RGL antibody, whereas the polyclonal antibodies did not recognize the 18 kDa (PE-1-immunoreactive) band. We propose that the immunological and size characteristics of some of these peptides (29, 24/22, 15 kDa) suggest their similarity to the peptides of predicted molecular mass 23·3, 18·2 and 12·6 kDa previously found in bovine adrenal medulla. The results also indicate the existence of high molecular weight pro-enk-A peptides shortened at the N-terminus. The use of an immunoradiometric assay designed to measure the proenk-A-derived 18·2 kDa peptide using PE-2 and an affinity purified and radioiodinated MetEnk-RGL IgG has supported these findings.

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