scholarly journals Characterization of the coupling activity for the binding of inter-alpha-trypsin inhibitor to hyaluronan in human and bovine follicular fluid

Reproduction ◽  
2002 ◽  
pp. 249-257 ◽  
Author(s):  
L ODum ◽  
CY Andersen ◽  
TE Jessen
Keyword(s):  
Trypsin Inhibitor ◽  
Follicular Fluid ◽  
Covalent Binding ◽  
Coupling Reaction ◽  
Heavy Chains ◽  
Cumulus Oocyte Complex ◽  
Rate Limiting Step

The plasma proteinase inter-alpha-trypsin inhibitor is necessary for normal expansion of the cumulus-oocyte complex (COC) and lack of inter-alpha-trypsin inhibitor results in severe infertility. After diffusion from the circulation into the follicles, inter-alpha-trypsin inhibitor is incorporated into the extracellular hyaluronan network of the expanding COC. However, mixing isolated inter-alpha-trypsin inhibitor with hyaluronan in vitro does not result in coupling to hyaluronan. Other components must be present. A recently developed electrophoretic technique by which hyaluronan-bound inter-alpha-trypsin inhibitor is immobilized was used to demonstrate coupling activity in human and bovine follicular fluid that is necessary for the formation of a firm binding between inter-alpha-trypsin inhibitor heavy chains and hyaluronan, as observed in vivo. No coupling activity could be detected in human serum. Coupling occurred only in the presence of follicular fluid. The coupling activity of follicular fluid was irreversibly destroyed by heat treatment, lowering of pH or tryptic digestion, indicating that the coupling activity is associated with a protein. Calcium ions are essential for the coupling reaction. The binding reaction in vitro using intact inter-alpha-trypsin inhibitor is slow and occurs over 24 h. The early-formed complexes between inter-alpha-trypsin inhibitor and hyaluronan contain small amounts of bikunin, whereas the end product contains heavy chains and essentially no bikunin. The heavy chains released from inter-alpha-trypsin inhibitor by NaOH treatment bound immediately to hyaluronan, indicating that the dissociation of heavy chains from inter-alpha-trypsin inhibitor is the rate-limiting step. In conclusion, at least four components are essential for the covalent binding of heavy chains to hyaluronan: inter-alpha-trypsin inhibitor and calcium from plasma, hyaluronan and one or more proteins found in follicular fluid.

Glycosaminoglycan-bound and free inter-α-trypsin inhibitor components of follicular fluid

Zygote ◽  
2001 ◽  
Vol 9 (4) ◽  
pp. 283-288 ◽  
Author(s):  
Lars Ødum ◽  
Torben E. Jessen ◽  
Claus Yding Andersen
Keyword(s):  
Extracellular Matrix ◽  
Trypsin Inhibitor ◽  
Follicular Fluid ◽  
Normal Female ◽  
Heavy Chains ◽  
Rate Limiting Step ◽  
Fluid Concentration ◽  
Rate Limiting

The proteinase inhibitor inter-α trypsin inhibitor (ITI) is a blood-derived protein necessary for normal female fertility. Absence of ITI leads to ovulation of naked oocytes that cannot fertilise. ITI consists of two heavy chains (ITI-HC) and bikunin linked by a chrondroitin sulphate. By binding to hyaluronate, ITI-HC stabilises the extracellular matrix, but ITI-HC also binds to proteoglycans in follicular fluid. In vivo concentrations of ITI components in preovulatory follicular fluid, free as well as bound to hyaluronate or proteoglycan, are unknown. In order to quantify these components, 58 follicular fluids and 13 blood samples were collected in connection with in vitro fertilisation and embryo transfer treatment of 13 women. Quantitation of glycosaminoglycan-bound ITI-HC was performed after separation from free ITI in agarose gel. ITI components were determined by immunoelectrophoresis and hyaluronate by an ELISA method. The follicular fluid concentration of ITI was on average 70% of that in plasma and the concentration of hyaluronate remained low despite follicular production, suggesting that the production of hyaluronate is the rate-limiting step in the formation of the extracellular matrix of the oocyte-cumulus complex. In follicular fluid, the concentration of free ITI-HC was higher than that of glycosaminoglycan-bound ITI-HC. Addition of exogeneous hyaluronate doubled the amount of hyaluronate-bound ITI-HC, further supporting the notion that ITI in follicular fluid is not rate-limiting for cumulus expansion in vivo.

scholarly journals Role of tumour necrosis factor stimulated gene 6 (TSG-6) in the coupling of inter-alpha-trypsin inhibitor to hyaluronan in human follicular fluid

Reproduction ◽  
2003 ◽  
pp. 27-31 ◽  
Author(s):  
TE Jessen ◽  
L Odum
Keyword(s):  
Tumour Necrosis Factor ◽  
Trypsin Inhibitor ◽  
Calcium Ions ◽  
Follicular Fluid ◽  
Tumour Necrosis ◽  
Coupling Reaction ◽  
Human Follicular Fluid ◽  
Heavy Chains ◽  
Necrosis Factor

Binding of the plasma proteinase inhibitor inter-alpha-trypsin inhibitor (ITI) to hyaluronan is necessary for normal expansion of the cumulus-oocyte complex. Lack of ITI causes severe infertility. Binding of ITI to hyaluronan depends on calcium ions and coupling activity present in follicular fluid (Odum et al., 2002). The complexes formed by this process contain ITI heavy chains bound to hyaluronan, and bikunin is detached from ITI during the coupling reaction. In the present study, an electrophoretic technique by which hyaluronan-bound ITI is immobilized was used to demonstrate that tumour necrosis factor stimulated gene 6 (TSG-6) is necessary for the coupling reaction. Thus, immunoprecipitation of TSG-6 in human follicular fluid eliminates the coupling reaction and re-addition restores the activity. However, it appears that components other than hyaluronan, ITI, calcium ions and TSG-6 are involved in the coupling reaction, as in vitro incubation of these components does not generate stable complexes between ITI heavy chains and hyaluronan unless some follicular fluid is added. In conclusion, TSG-6 is necessary for the coupling of ITI to hyaluronan, but at least one additional component in follicular fluid is essential.

Inhibition of LH-stimulated cyclic AMP formation in pre-ovulatory rat granulosa cells by follicular fluid

1980 ◽  
Vol 95 (1) ◽  
pp. 84-89 ◽  
Author(s):  
Knut Nordenström ◽  
Anita Sjögren ◽  
Lars Hamberger
Keyword(s):  
Granulosa Cells ◽  
Follicular Fluid ◽  
Phosphodiesterase Inhibitor ◽  
Inhibitory Effect ◽  
Female Rats ◽  
Bicarbonate Buffer ◽  
Ovulatory Follicles

Abstract. Immature female rats were injected sc with a single dose of PMSG to induce growth and maturation of ovarian follicles. In the morning of prooestrus the rats were given a single ip injection of LH (10 μg/rat) or 0.154 m NaCl, 2 h prior to sacrifice. Granulosa cells were isolated from the pre-ovulatory follicles and incubated in Krebs bicarbonate buffer, for 1 h with or without in vitro addition of various test substances. Following incubation the amounts of cAMP in tissue plus medium were determined. It was found that the isolated granulosa cells exposed to LH in vivo responded to the addition of LH in vitro with a production of high amounts of cAMP, i.e. these cells were not refractory to LH stimulation and in fact responded better than granulosa cells isolated from ovaries not exposed to LH in vivo. The addition to the incubation medium of follicular fluid (FFl) obtained from pre-ovulatory follicles decreased the effect of LH in vitro when added at a final concentration of 1% and completely abolished it at a concentration of 3%. Removal of steroids from the FFl did not influence the inhibitory effect and the addition of a phosphodiesterase inhibitor (IBMX) in vitro did not alter the results in principle. These results point to the existence of a factor in the FF1 which interacts with the sensitivity of the isolated preovulatory granulosa cells to repeated exposures to LH. Characterization of this factor is subject to further investigations.

scholarly journals Characterization of Cdc47p-minichromosome maintenance complexes in Saccharomyces cerevisiae: identification of Cdc45p as a subunit.

1997 ◽  
Vol 17 (10) ◽  
pp. 5867-5875 ◽  
Author(s):  
S Dalton ◽  
B Hopwood
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Replication ◽  
Chromosomal Dna ◽  
Minichromosome Maintenance ◽  
Rate Limiting Step ◽  
A Subunit

Cdc47p is a member of the minichromosome maintenance (MCM) family of polypeptides, which have a role in the early stages of chromosomal DNA replication. Here, we show that Cdc47p assembles into stable complexes with two other members of the MCM family, Cdc46p and Mcm3p. The assembly of Cdc47p into complexes with Cdc46p does not appear to be cell cycle regulated, making it unlikely that these interactions per se are a rate-limiting step in the control of S phase. Cdc45p is also shown to interact with Cdc47p in vivo and to be a component of high-molecular-weight MCM complexes in cell lysates. Like MCM polypeptides, Cdc45p is essential for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae; however, Cdc45p remains in the nucleus throughout the cell cycle, whereas MCMs are nuclear only during G1. We characterize two mutations in CDC47 and CDC46 which arrest cells with unduplicated DNA as a result of single base substitutions. The corresponding amino acid substitutions in Cdc46p and Cdc47p severely reduce the ability of these polypeptides to assemble in a complex with each other in vivo and in vitro. This argues that assembly of Cdc47p into complexes with other MCM polypeptides is important for its role in the initiation of chromosomal DNA replication.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

Evaluation of the In Vivo Acute Toxicity and In Vitro Hemolytic and Immunomodulatory Activities of the Moringa oleifera Flower Trypsin Inhibitor (MoFTI)

2020 ◽  
Vol 27 ◽  
Author(s):  
Leydianne Leite de Siqueira Patriota ◽  
Dayane Kelly Dias do Nascimento Santos ◽  
Bárbara Rafaela da Silva Barros ◽  
Lethícia Maria de Souza Aguiar ◽  
Yasmym Araújo Silva ◽  
...  
Keyword(s):  
Acute Toxicity ◽  
Trypsin Inhibitor ◽  
Hemolytic Activity ◽  
Moringa Oleifera ◽  
Biological Activities ◽  
Hematological Parameters ◽  
Behavioral Alterations ◽  
Female Mice

Background: Protease inhibitors have been isolated from plants and present several biological activities, including immunomod-ulatory action. Objective: This work aimed to evaluate a Moringa oleifera flower trypsin inhibitor (MoFTI) for acute toxicity in mice, hemolytic activity on mice erythrocytes and immunomodulatory effects on mice splenocytes. Methods: The acute toxicity was evaluated using Swiss female mice that received a single dose of the vehicle control or MoFTI (300 mg/kg, i.p.). Behavioral alterations were observed 15–240 min after administration, and survival, weight gain, and water and food consumption were analyzed daily. Organ weights and hematological parameters were analyzed after 14 days. Hemolytic activity of MoFTI was tested using Swiss female mice erythrocytes. Splenocytes obtained from BALB/c mice were cultured in the absence or presence of MoFTI for the evaluation of cell viability and proliferation. Mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) levels were also determined. Furthermore, the culture supernatants were analyzed for the presence of cytokines and nitric oxide (NO). Results: MoFTI did not cause death or any adverse effects on the mice except for abdominal contortions at 15–30 min after administration. MoFTI did not exhibit a significant hemolytic effect. In addition, MoFTI did not induce apoptosis or necrosis in splenocytes and had no effect on cell proliferation. Increases in cytosolic and mitochondrial ROS release, as well as ΔΨm reduction, were observed in MoFTI-treated cells. MoFTI was observed to induce TNF-α, IFN-γ, IL-6, IL-10, and NO release. Conclusion: These results contribute to the ongoing evaluation of the antitumor potential of MoFTI and its effects on other immunological targets.

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