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S Cseh

Publications and source records attributed to S Cseh.

28 records · Page 2Linked to original sources
PubMed1996

Rapid freezing of mouse embryos in ethylene glycol at different preimplantation stages.

The objectives of this study were to examine the effects of a rapid freezing protocol on the survival and in vitro development of mouse embryos cryopreserved in ethylene glycol (EG) at different preimplantation stages, and secondly, to investigate the effect of exposure to 3.0 M EG with 0.25 M sucrose on the survival and in vitro development of mouse embryos without freezing at different developmental stages. To perform the rapid freezing procedure, embryos were equilibrated in Dulbecco's phosphate buffered saline (DPBS) containing 3.0 M EG and 0.25 M sucrose (freeze medium) for 20 min and loaded into 250 microliters straws in a single column of freeze medium. The straws were held in liquid nitrogen (LN2) vapour for 2 min and immersed into LN2. Embryos were thawed in a 37 degrees C water bath for 20 sec and transferred to DPBS supplemented with 0.5 M sucrose (rehydration medium) for 10 min and cultured for 24 to 96 h in HTF (Human Tubal Fluid) plus 4 mg/ml BSA (Bovine Serum Albumin). Significant differences were found in the survival and development of mouse embryos at different developmental stages rapid frozen in EG and sucrose: two cell 43/84 (51%), 4-8 cell 44/94 (47%), morula and early blastocyst 56/70 (80%), expanding and expanded blastocysts 10/59 (17% (p < 0.05). These data indicate that the developmental stage in which mouse embryos are subjected to this quick freeze protocol affects survival and development in vitro and the majority (80%) of morula and early blastocyst stage embryos survive the procedure. No significant differences were observed in the in vitro developmental capacity of embryos at different developmental stages after treatment with high concentrations (3.0 M) of EG solution without freezing. Further investigations are underway to better understand the reasons for different survival rates of embryos frozen at different developmental stages using the present procedure.

Animals↗
PubMed1994

The structure and function of the first component of complement: genetic engineering approach (a review).

The availability of cDNA and genomic clones for the subcomponents of C1, as well as the recognition of the modular organization of serine-proteases have opened up exciting new possibilities for approaching structural problems. In this review the latest achievements of combined protein engineering, functional and structural studies are summarized. The concept of this research is to construct deletion, point and hybrid mutants of the highly homologous C1r and C1s subcomponents, to reveal the functional role of individual modules, map the interaction sites between subcomponents of the C1 complex and refine the structural model of C1. The first prerequisite of such an approach was the expression of the subcomponents in a eukaryotic system, in biologically active form. This was followed by expression of various mutants. Autographa californica nuclear polyhedrosis virus was used as vector to express human C1r and C1s in Spodoptera frugiperda cell culture and in lepidopteran larvae. The yield of expression was high enough to isolate recombinant subcomponents for structural and functional studies. Recombinant viruses containing the A-, B-, and C-chains of C1q were also constructed. The insect cells are able to beta-hydroxylate the Asn residue of the EGF domain in the C1r but with a low efficiency. It is clear now, that this post-translational modification does not play a role in the Ca2+ dependent C1r-C1s interaction. The results with deletion mutants of C1r show that both, domain I, and II are absolutely necessary for the tetramer formation and both have regulatory role in the autoactivation. The C1s alpha R hybrid does not dimerize in presence of Ca2+, however it can form a tetramer with C11(2) that can bind to C1q. This observation indicates that the function of the C1s alpha part in the hybrid is modulated by the C1r part (gamma B) of the molecule. The C1Rs hybrid behaves like C1r, providing haemolytically active C1 with C1q and C1s. This observations shows that the regulatory domains determine the high functional specificity of the serine-protease subcomponents of C1. In order to control the autoactivation process point mutant cDNAs were constructed by altering the Arg-Ile bond in the catalytic domain of the C1r. The Gln-Ile construction is a stable zymogen while the Arg-Phe mutant has a lower rate of autoactivation.

Animals↗
PubMed1993

Protein engineering studies on C1r and C1s.

1. C1r and C1s cDNAs were placed downstream the strong polyhedrin promoter in the Autographa californica nuclear polyhedrosis virus and the recombinant proteins were expressed in insect cells, in biologically active form. The yield of expression is high enough to get recombinant components for chemical and functional studies (5 micrograms/ml cell culture supernatant). 2. The biological activity and the post-translational modifications of the recombinant subcomponents were checked. The rC1r and rC1s proved to be biologically active in the hemolytic assay, although their glycosylations were different compared to that of the serum proteins. The insect cells are able to beta-hydroxylate the Asn residue of the EGF domain in the C1r but with a low efficiency. It is clear now, that this post-translational modification does not play a role in the Ca2+ dependent C1r-C1s interaction. 3. Two deletion mutants of C1r cDNA were constructed in order to clarify the role of domain I and II. The results show that both, domain I, and II are absolutely necessary for the tetramer formation and both have a regulatory role in the autoactivation. The autoactivation of the mutants is accelerated significantly. 4. Hybrid cDNA constructions were also made, and one of them was expressed. In the C1s alpha R hybrid the C1s alpha part cannot dimerize in presence of Ca2+, but it can form a tetramer with C1r2, that can bind to C1q. This observation indicates that the function of the C1s alpha part in the hybrid is modulated by the C1r part (gamma B) of the molecule. 5. In order to control the autoactivation process point mutant cDNAs were constructed through altering the Arg-Ile bond in the catalytic domain of the C1r. The Gln-Ile construction is a stable zymogen while the Arg-Phe mutant has a lower rate of autoactivation. These results do justify our approach of using domain-domain interchange, domain deletion and point mutations in combination, to reveal the structural background of C1 function at intramolecular level.

Animals↗
PubMedDate not supplied

Functional effects of domain deletions in a multidomain serine protease, C1r.

The C1r subcomponent of the first component of complement is a complex, multidomain glycoprotein containing five regulatory or binding modules in addition to the serine protease domain. To reveal the functional role of the N-terminal regulatory domains, two deletion mutants of C1r were constructed. One mutant comprises the N-terminal half of domain I joined to the second half of the highly homologous domain III, resulting in one chimeric domain in the N-terminal region, instead of domains I-III. In the second mutant most of the N-terminal portion of domain I was deleted. Both deletion mutants were expressed in the baculovirus-insect cell expression system with yields typical of wild type C1r. Both mutants maintained the ability of the wild type C1r to dimerize. The folding and secretion of the recombinant proteins was not affected by these deletions, and C1-inhibitor binding was not impaired. The stability of the zymogen was significantly decreased however, indicating that the N-terminal region of the C1r molecule contains essential elements involved in the control of activation of the serine protease module. Tetramer formation with C1s in the presence of Ca2+ was abolished by both deletions. We suggest that the first domain of C1r is essential for tetramer formation, since the deletion of domain I from C1r impairs this interaction.

Amino Acid Sequence↗
PubMedDate not supplied

Controlled ovarian stimulation and ultrasound guided follicular aspiration in the baboon (Papio cynocephalus anubis).

The objective of this study was to investigate whether baboon females respond to an ovarian stimulation protocol incorporating pituitary suppression with a GnRH agonist (GnRHa) and highly purified human FSH (hphFSH) with follicular development and oocyte maturation. An ovulation induction protocol was applied to 5 adult female baboons with a history of regular menstrual cycles (33-34 days). A long-acting GnRHa implant containing goserelin acetate was placed s.c. on days 22-24 of their menstrual cycle. Daily hphFSH (75 IU im) treatments were started approximately 10 days following menses. When the majority of the follicles were > or = 5 mm in diameter and the E2 levels had reached a maximum, hCG (2000 IU i.m.) was administered to induce final maturation of the oocytes and ovulation. 30 to 34 h after hCG administration, transabdominal follicular aspiration was performed using a variable frequency transvaginal transducer with ultrasound. A total of 71 oocytes were collected (average: 17). 91% of the oocytes were morphologically normal indicating that they were appropriate for in vitro insemination.

Animals↗