Biomedical subjects

M Sárvári

Publications and source records attributed to M Sárvári.

6 recordsLinked to original sources

PubMed2003

Inhibition of C1q-beta-amyloid binding protects hippocampal cells against complement mediated toxicity.

Activation of complement by beta-amyloid (A beta) contributes to the pathology of Alzheimer's disease (AD). Here, we show that C1-Inhibitor (C1-Inh) protects cultured rat hippocampal cells against beta-amyloid induced complement lysis indicating a classical pathway-mediated activation mechanism. We report on screening of compound libraries to identify compounds that inhibit C1q binding to beta-amyloid. Characterization of these compounds revealed that C1q possessed distinct binding sites for beta-amyloid and antibodies. One selected compound protected cultured hippocampal cells against complement-dependent beta-amyloid toxicity. These results provide evidence that complement has the potential to damage hippocampal cells, and C1q is a relevant target to suspend this deleterious mechanism in Alzheimer's disease.

Amyloid beta-Peptides↗

PubMed1997

Baculovirus-mediated trans-epithelial transport of proteins in infected caterpillars.

Baculovirus-mediated abnormal protein trafficking was studied in infected caterpillars by using heterologous proteins. The gene for human complement C1r was expressed in larvae of Mamestra brassicae by a recombinant Autographa californica nuclear polyhedrosis virus (AcMNPV) vector. By following the time-course of recombinant C1r distribution among various tissues, cell types and cell organelles, we concluded that the dominant site of recombinant protein synthesis was the fat body, although some production in the haemocytes and midgut was also observed. Only about 4% of the cells of the infected organs expressed recombinant C1r, which was then secreted into the haemolymph. The tracheal and integumental cuticle was rich in recombinant protein from the fourth day after infection although epidermal cells did not synthesize recombinant C1r. The morphological picture suggested that the accumulation was a consequence of a trans-epithelial transport. This transport process was checked by following the fate of the 49 kDa haemolymph protein and injected ovalbumin in AcMNPV-infected Mamestra brassicae and in Lymantria dispar nuclear polyhedrosis virus-infected Lymantria dispar larvae. Both proteins were able to pass the basal membrane of the epidermis and accumulated in the cuticle, while in control larvae neither was transported. The observed trans-epithelial transport points to the role of baculoviruses in directing recombinant, endo- and exo-genous proteins to cuticulated tissues. Based on these results we conclude that the permeability of basal membranes undergoes a characteristic change during the course of baculovirus infection.

Animals↗

PubMed1990

Ecdysteroids increase the yield of recombinant protein produced in baculovirus insect cell expression system.

Spodoptera frugiperda cells are the hosts of wild type and recombinant virus in the baculovirus insect cell expression system. The expression of the foreign gene could be enhanced by the addition of ecdysteroids and increased amount of recombinant protein was secreted into the medium. The time and concentration dependence of this effect was followed in the case of 20-hydroxyecdysone-, makisterone and ecdysone. 20-hydroxyecdysone proved to be the most efficient, producing a three fold increase in the level of recombinant protein secreted into the medium, as it was measured by ELISA. This effect was also confirmed by tracing the L-(35S)methionine incorporation into the gene product. Makisterone was also effective in stimulation, while ecdysone proved to be ineffective.

Animals↗

PubMed1989

Expression of hemolytically active human complement component C1r proenzyme in insect cells using a baculovirus vector.

The gene of human C1r has been expressed in a baculovirus-insect-cell system via the pAc373 transplacement vector. The full-length cDNA copy was inserted into the pAc373 vector downstream from the strong polyhedrin promoter of the baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV). Spodoptera frugiperda cells were cotransfected with the resultant plasmid, pAcC1r, and the wild-type AcNPV DNA. Recombinant viruses, which drove the expression of C1r protein, were selected by plaque morphology and ELISA. Insect cells infected with the recombinant virus produced and secreted human C1r protein, at a level of 1-2 mg/l of medium. The expressed C1r was isolated from the medium by chromatofocusing. On reducing gels only a single Coomassie-staining band was observed, and this band migrated at 80-83 kD characteristic of the unactivated C1r proenzyme. Its identification as C1r was immunologically confirmed on Western blots. C1 reconstituted from purified C1r expressed in insect cells together with human C1q and C1s proved biologically active in a hemolytic assay. Thus, the baculovirus-insect-cell system is capable of expressing and secreting a sophisticated, multifunctional human complement subcomponent in its biologically activatable form.

Animals↗

PubMed1987

A 'molten globule'-like unfolding intermediate of a four domain protein, the Fc fragment of the IgG molecule.

The Fc fragment of human IgG1 can be trapped in a stable intermediate state during thermal denaturation. In this conformation the molecule is compact with a native-like secondary structure, however, the tertiary structure is perturbed as revealed by intrinsic fluorescence measurements, the near-UV CD spectra and by mapping of antigenic sites with monoclonal antibodies. Similar phenomena were recently described for a few globular proteins of small size, and termed 'the molten globule' state. Our observation is a unique example of this phenomenon for a four domain protein.

Antibodies, Monoclonal↗

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↗